In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn The envelope glycoprotein gp120-gp41 complex of HIV-1 mediates receptor attachment and virus-cell membrane fusion, leading to cellular entry. A shield of asparagine-linked oligosaccharides occludes the gp120-gp41 protein surface and evolution of this glycan shield provides a means for evading circulating neutralizing antibody. Here we examined how conserved structural elements of the glycoprotein complex, in particular the gp120-gp41 association site, retain functionality in the context of glycan shield evolution. This information is important for the evaluation and exploitation of such conserved functional determinants as potential drug and/or vaccine targets. Our data indicate that the loss of either of 2 adjacent glycans in variable region 1 of gp120 leads to changes in local and remote glycan-dependent epitopes and that this is linked to a remodelling of gp120-gp41 interactions in order to maintain a functional gp120-gp41 complex. We propose that this represents a mechanism for the functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of neutralizing antibody selection. The HIV-1 envelope glycoprotein (Env) complex comprises a trimer of gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits and mediates viral attachment, membrane fusion and viral entry  that causes defective gp120-gp41 association. The defective phenotype was restored by second site mutations leading to the loss of PNGSs at V1 positions 136 and 142, the former operating in conjunction with L494I in C5 of gp120 and the latter requiring D601N in the DSR. The PNGS mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on LeuAD8-K601D to serial PBMC passage in order to select suppressor mutations. We reasoned that the locations of suppressors would point to structural elements that are functionally linked to the DSR, thereby shedding light on its role in Env function. We first confirmed that the K601D mutation promoted the shedding of gp120 into the culture supernatant of 293T cells transfected with pcDNA3.1-AD8env, as determined by radioimmunoprecipitation  from independent donors was severely attenuated by the mutation .In this study, we subjected the gp120-gp41 association defective DSR mutant HIV-1pitation Fig. 1C.env region was PCR-amplified from genomic DNA isolated from infected cells at days 20, 30, 40 and 50 for culture P0, and days 30 and 50 for culture P2. The PCR products were cloned into the pΔKAD8env expression vector for DNA sequencing. The K601D mutation was retained in P0 clones isolated at days 20 and 30 (136VT) within V1 of gp120 (3/6 clones). By day 50, when mutant viral replication approached that of WT, the T138N/L494I/K601N triple mutation was present in 42% of clones. A single clone (P0.D50.10) contained S144N, which ablates the nearby PNGS at N142SS.In order to identify suppressors of K601D, the 0 and 30 , when re0 and 30 , P0. The, P2). Deletion of residues N139INN (ΔN139INN) from V1, that results in the loss of overlapping PNGSs at positions 141 and 142 (N141N142SS), was observed in 3/14 P2 day-30 clones, while 2/14 possessed mutations at Thr138; K601N was also observed in 50% of P2-day 30 clones , which also ablates the overlapping PNGSs at 141 and 142. Interestingly, the L494I mutation was not observed in P2 clones. These data indicate that PNGSs in V1 are functionally linked to the gp120-gp41 association synapse.In an independent PBMC long-term culture (P2), the replication competence of the mutant virus approached that of WT by the 3rd passage . The T138N/L494I/K601D and T138N/L494I/K601N genotypes exhibited near-WT replication kinetics in PBMCs from both donors. These data suggest that T138N, which ablates the PNGS at Asn136 within V1, and L494I act synergistically to suppress K601D (and K601N). The analysis of P2 genotypes in PBMC cultures indicated that the N139INN deletion in V1 was not sufficient to suppress K601D, but its combination with K601N led to near-WT levels of viral replication .To determine how replication was restored to the K601D virus in the long-term PBMC cultures, we reconstructed the dominant P0 and P2 genotypes in the context of the pAD8 proviral clone and examined viral replication in two independent PBMC donors. The P0 mutations, L494I within C5 and K601N within the DSR, partially restored replication in the PBMCs of one donor but not in the second, apparently less permissive donor . These data indicate that the K601D and K601N DSR mutations inhibit cell-cell fusion function by destabilizing the gp120-gp41 complex. However, the effect of K601N on Env function in the context of virus replication appears to be donor dependent (see 136 (The pΔKAD8139INN/K601D and ΔN139INN/K601N P2 clones exhibited 74–77% of WT fusion activity (P<0.05) with only partial restoration of gp120-gp41 association, even though only the latter clone was competent for replication in PBMCs . The suppression of the K601N fusion and replication phenotype by ΔN139INN is therefore not dependent on the full restoration of gp120-gp41 association or on the L494I C5 mutation. gp120 molecules with the ΔN139INN mutation migrated faster in SDS-PAGE than non-ΔN139INN-containing Envs, again consistent with the loss of glycan , although the position varies. Combining S144N (a PNGS mutation observed in clone P0.D50.10) with K601N resulted in almost identical fusion activity to ΔN139INN/K601N . These data are consistent with homogeneous oligomannose type Env glycosylation due to the action of kifunensine 139INN/K601N revertant Envs. Figure S1B shows that the presence of kifunensine leads to a general elevation in the fusion activities of the 4 constructs tested. Importantly, the presence of kifunensine did not affect the relative fusion activities of the T138N/L494I/K601N and ΔN139INN/K601N revertant Envs with respect to wild type and K601D. These data were reflected in the gp120-gp41 association assay where the shedding phenotype of K601D was restored to wild type and near-wild type levels, respectively, in T138N/L494I/K601N and ΔN139INN/K601N, in both the presence and absence of kifunensine . Overall, these data indicate that the fusion and gp120-gp41 association phenotypes of the cell-expressed revertant Envs are not influenced by the presence of complex or hybrid-type glycans.Scanlan and coworkers 139INN do not increase the cell-cell fusion activity of surface expressed Env or the infectivity of Env-pseudotyped luciferase reporter virus when introduced to the WT background. We also noted that the L494I mutation in a WT background did not substantially increase cell-cell fusion or gp120-gp41 association (data not shown). These data are consistent with specific functional crosstalk between the V1 glycans, the Asp601 or Asn601 in the DSR and position 494 in C5.We next asked whether the V1 PNGS mutations restored function via a functional link to Asp or Asn at position 601 in the DSR or whether a generalized enhancement in Env function could explain the restored phenotypes. env vectors and determined their effects on cell-cell fusion, glycoprotein processing and gp120-gp41 association. env-transfected 293T cell lysates with the gp41-specific mAb, C8, confirmed that the mutant glycoproteins were expressed and processed to gp41 at levels that were close to the WT  , H88A , K171A, E172A and Q188A , and F264A and R274A  CCR5 mutants supported fusion with the 3 Env constructs to the same extent as WT CCR5, whereas fusion with Q280A (extracellular loop 3) was decreased to 25–40% of WT CCR5 activity. We also compared the abilities of T138N/L494I/K601N, ΔN139INN/K601N and WT Envs to mediate cell-cell fusion with the CCR5-Y14N tyrosine sulfation mutant, which exhibits a lower affinity for gp120-gp41 and functions as a HIV-1 coreceptor in a cell surface concentration-dependent manner luc vector and a dilution series of pc.CCR5 DNA; the fusion activity of ΔN139INN/K601N was slightly diminished across the pc.CCR5 dilution series  . 139INN deletion did not provide any improvement to L593V fusogenicity, but restored increasing levels of fusion function to K601D and W596L, respectively. These data indicate that T138N in V1 alters the gp120-gp41 association site such that fusion function is less dependent on Trp596 and, when L494I is also present, on Leu593 and Lys601. The N139INN deletion renders fusion function less dependent on Trp596 and Lys601.The DSR mediates association with gp120 and may play a role in the activation of gp41 by responding to receptor-induced changes in gp120 139INN mutations were linked to changes in the neutralization sensitivity of Env-pseudotyped reporter viruses. The results . By contrast, neutralization by IgGb12  indicating that these glycans are likely to modulate neutralization epitopes recognized by human immune sera. These data indicate that the adjacent V1 glycans shown here to modulate the gp120-gp41 association site are linked to neutralization resistance.Previous studies have shown that larger numbers of potential N-linked glycans in V1V2 correlate with resistance to NAb  results Fig. 7 snd site mutations that suppress the defect will point to structural elements within gp120-gp41 with functional links to the DSR. The loss of glycans at Asn136 or Asn142 within V1, the former acting in conjunction with L494I in C5 and the latter with a D601N pseudoreversion, largely suppressed the functional defects, with Env function becoming less dependent on particular gp120-contact residues  within the DSR.The gp120-gp41 complex of HIV-1 is maintained through interactions between C1 and C5 of gp120 with the DSR of gp41. Receptor engagement elicits a conformational signal that is transmitted through gp120 and sensed by the gp120-contact residues to activate the membrane fusion potential of gp41. In this study, we forced the evolution of a DSR mutant (K601D) with defective gp120-gp41 association, reasoning that the locations of emergent 2601, but a WT-like phenotype also required deletion of the glycan at Asn136 in V1 through T138N. Outgrowth by Asn601-containing clones suggested that the negative charge of Asp601 was not favoured in the long term. Cell-cell fusion and viral replication were also restored by a short ΔN139INN deletion in V1 operating in conjunction with a D601N pseudoreversion. In this case Env fusogenicity was maintained despite a more labile gp120-gp41 complex. The ΔN139INN/K601N phenotype was largely recapitulated by S144N/K601N, suggesting that loss of the Asn142 glycan is sufficient to restore fusion function when Asn601 is present in the DSR. Mutations designed to ablate the remaining PNGSs at positions 141, 156, 160 and 186 within V1V2, did not restore function to Asn601-containing Envs, indicating a specific functional linkage between the DSR and the Asn136 and Asn142 glycans in the context of AD8 Env. It should be noted that V1V2 exhibits high degree of diversity in sequence, length and numbers of PNGSs One mechanism of reversion involved the acquisition of L494I in C5 that led to slightly improved gp120-gp41 association and fusion function with Asp139INN with various DSR mutations: L593V and K601D, which disrupt gp120-gp41 association and fusion function, and W596L which inhibits fusion 593 and Lys601, whereas less dependence on Lys601 was observed with ΔN139INN. In the case of W596L, T138N and ΔN139INN were sufficient to confer WT fusion levels, but this did not involve changes to the WT-like gp120-gp41 association phenotype of W596L. The fusion gains conferred to W596L may involve transduction of a receptor-induced activation signal from gp120 to gp41 through the association site in manner that is less dependent on Trp596. The earlier finding that W596L blocks sCD4-induced formation of the gp41 prehairpin intermediate indicates that Trp596 can play a role in receptor-triggered gp41 activation 591, Gly597, Thr606 and Trp610The impacts of the V1 glycan changes on the gp120-gp41 association site were probed further by combining T138N, L494I and ΔN126-Cys196 and Cys131-Cys157). The highly variable segments and PNGSs are for the most part contained within the connecting loops  and remote (2G12) glycan-dependent neutralization epitopes as well as the global antigenic structure of Env (HIVIG) and that these changes are functionally linked to a remodelling of the gp120-gp41 association site. Our data also imply that glycan shield evolution may indirectly affect the inhibitory potential of novel fusion blockers, such as PF-68742, for which the DSR is a component of their targeting mechanism A striking feature of gp120-gp41 is the occlusion of the conserved protein surface by a glycan shield comprising ∼24 N-linked glycans on gp120 and 4–5 on gp41 593, Trp596 and Lys601) less important for gp120 association and fusion function, and thereby implying that gp120-gp41 contact residues at alternative positions within the synapse become utilized for these functions. The allosteric modulation of the conserved DSR-C1–C5 synapse by distal V1 glycans may represent a mechanism whereby functionally relevant gp120-gp41 association is maintained as the virus acquires neutralization resistance due to the evolution of its glycan shield.Our previous work indicated that the gp120-gp41 association interface is structurally and functionally plastic despite exhibiting a high degree of sequence conservation AD8 Env expression vector, pCDNA3.1-AD8env, is described elsewhere env was derived by religation of the end-filled HindIII and EcoRI sites of pCDNA3.1-AD8env. In vitro mutagenesis of the gp41 region was carried out by overlap extension PCR. Mutants of the pAD8 infectious clone . The supernatants were assayed for RT activity at various time points.PBMC infections were conducted as described previously AD8 (according to RT activity) in parallel and maintained in culture for 10 days. Cell-free culture supernatants were filtered (0.45 µm pore size) and normalized according to RT activity prior to the next passage . Genomic DNA was extracted from infected PBMCs using Qiagen DNeasy. The viral DNA fragment encompassed by nucleotides 5954–9096 (HXB2R numbering convention) was PCR-amplified using Expand HiFi (Roche) and the primers, 5′-GGCTTAGGCATCTCCTATGGCAGGAAGAA (Env1A) and 5′-TAGCCCTTCCAGTCCCCCCTTTTCTTTTA (Env1M) env (KpnI-XbaI) and the entire env open reading frame sequenced using ABI BigDye terminator 3.1.Phytohemagglutinin-stimulated PBMCs were infected with equivalent amounts of wild type (WT) and K601D-mutated HIV-1Lysates of Env-expressing 293T cells or virions pelleted from pAD8-transfected 293T cell supernatants were subjected to SDS-PAGE under reducing conditions, transferred to nitrocellulose and then probed with mAb C8 to gp41 , and then labelled for 45 min with 150 µCi Tran-35S-label . The cells were then washed and chased in complete medium for 5–6 h prior to lysis. Cell lysates and clarified culture supernatants were immunoprecipitated with IgG14 or HIVIG and protein G Sepharose and subjected to SDS-PAGE in the presence of ß-mercaptoethanol 293T cells were transfected with pCDNA3.1-AD8To determine the effects of the competitive α-mannosidase inhibitor, kifunensine (Sigma), on Env synthesis and processing, cell-cell fusion and gp120-gp41 association, the compound was added to 293T cells at the time of transfection, and was maintained at the concentrations indicated in the results section through each step of the assays described above.gp120 and pCAG-T7 vectors using Fugene 6. At 24-h posttransfection, the cells were incubated for 30 min in cysteine and methionine-deficient medium, labelled for 45 min with 150 µCi Tran-35S-label, and then washed and chased in complete medium for 6 h. The clarified culture supernatants were adjusted to 0.6 M KCl, 1 mM EDTA and 1% w/v Triton-X100 and the gp120 content quantified following immunoprecipitation with IgG14 and protein G Sepharose, reducing SDS-PAGE and scanning in a Fuji phosphorimager. Equivalent amounts of WT and mutant gp120 proteins were incubated with serial dilutions of sCD4  and then incubated with mAb OKT4 and BSA-Sepharose on a vibrating platform . After pelleting the BSA-Sepharose, protein complexes were coprecipitated using protein G-Sepharose and gp120 quantified following reducing SDS-PAGE and scanning in a Fuji phosphorimager.293T cells were cotransfected with pTM-AD8139INN/K601N virus stocks were adjusted such that ∼2×106 relative light units (RLU) were obtained following a 48 h-infection of TZM-bl cells, a HeLa cell line expressing CD4 and CCR5 and harbouring integrated copies of the luciferase and β-galactosidase genes under control of the HIV-1 promoter . The cells were lysed 48 later and assayed for luciferase activity (Promega).Wild type, T138N/L494I/K601N and ΔN5 RLU following infection of U87.CD4.CCR5 cells was mixed with an equal volume of serially diluted IgG and incubated for 1 h at 37°C. One hundred microliters of the virus-IgG mixtures was then added to U87.CD4.CCR5 cells  and incubated for 2 days prior to lysis and assay for luciferase activity . Neutralizing activities for antibody samples were measured in triplicate and reported as the average percent luciferase activity. Purified IgG of monoclonal NAbs 2F5, 2G12 and IgGb12 were obtained from Polymun Scientific (Austria), PG16 was obtained from the International AIDS Vaccine Initiative, while HIVIG was obtained from F. Prince through the NIAID AIDS Research and Reference Reagent Program.Neutralization assays were conducted using a modification of the method of Dhillon et al. AD8 V1V2 was generated from PDB entry 3U4E in silico with oligomannose side chains using the glycosciences.de server (http://www.glycosciences.de/modeling/glyprot/php/main.php) A homology-based model of HIV-1Figure S1A, Western blot. Kifunensine was added to 293T cells at the time of transfection with ΔKADenv expression vectors. The transfected cells were cultured in the presence and absence of kifunensine for a further 48 h prior to lysis, reducing SDS-PAGE and Western blotting with DV-012 to gp120 (top panel) or 2F5 to gp41 (bottom panel). B, Cell-cell fusion. 293T cells were cotransfected with ΔKADenv expression vectors plus pCAG-T7 and cultured in the presence or absence of 10 µM kifunensine. At 24 h posttransfection, the cells were detached and cocultured for a further 18 h with pT4luc plus pcCCR5-cotransfected BHK-21 cells in the presence or absence of 10 µM kifunensine prior to lysis and assay for luciferase activity. ** P<0.01; **, P<0.001; T138N/L494I/K601N and ΔNINN/K601N versus K601D at the same kifunensine concentration; 2-tailed unpaired t test assuming unequal variances (n = 5). C, gp120-gp41 association. Kifunensine was added to 293T cells at the time of transfection with ΔKADenv expression vectors. At 24 h posttransfection, the cells were pulse-chase metabolically labelled in the presence or absence of 10 µM kifunensine prior to immunoprecipitation of culture supernatants (s) and cell lysates (c) with HIVIG, reducing SDS-PAGE and phosphorimager scanning.Effect of kifunensine on Env synthesis and function. (TIF)Click here for additional data file.Table S1Summary of cell-cell fusion, gp120-gp41 association and replication characteristics of revertant Env clones.(DOCX)Click here for additional data file.


H17 cells are widely regarded as the drivers of experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple sclerosis (MS). The factors, responsible for the generation and maintenance of TH17 cells as well as their participation in the pathogenic cascade leading to the demyelinating disease, have been studied extensively. However, how these harmful autoreactive cells are controlled in vivo remains unclear. By comparing TCR transgenic mice on a disease susceptible and a disease resistant genetic background, we show here that pathogenic TH17 cells are sequestered within the intestine of spontaneous EAE resistant B10.S mice. Disease resistant B10.S mice harbored higher frequencies of TH17 cells in the intestine compared to EAE susceptible SJL/J mice. Moreover, transferred TH17 cells selectively migrated to intestinal lymphoid organs of B10.S mice. The sequestration of TH17 cells in the gut was partially dependent on the gut homing receptor α4β7-mediated adhesion to the intestine. Administration of α4β7 blocking-antibodies increased the peripheral availability of TH17 cells, resulting in increased EAE severity after immunization in B10.S mice. Together, these results support the concept that the intestine is a check-point for controlling pathogenic, organ-specific T cells.Myelin-specific, pro-inflammatory T Evidence suggests that autoreactive T cells are commonly present in the healthy immune repertoire, but are kept in check by numerous tolerance mechanisms. Although several details of the tolerance mechanisms have yet to be elucidated, mechanisms including negative selection of autoreactive T cells in the thymus, ignorance, anergy, cytokine immune deviation, tolerogenic antigen presenting cells and induction of regulatory cells have been demonstrated to be involved in mediating self-tolerance H1 and IL-17-producing TH17 cells, either alone or in combination are capable of mediating a neurological disease in animals, resembling the human disease H17 cells in EAE as well as MS, due to their close association with several other autoimmune diseases H17 cells are crucial for controlling the invasion of pathogenic microorganisms and play an important role in intestinal immune homeostasis H17 cells induced neurological disease, supporting the idea that this T helper cell subset plays an important role in EAE pathogenesis H17 cells secrete IL-17a, IL-17f, IL-21 and IL-22 as their key effector cytokines and make use of the chemokine receptor CCR6 to enter the target tissues In vitro as well as in vivo generation of TH17 cells requires the induction of their master transcription factor retinoic acid-related orphan receptor-γt (ROR-γt) H17 cells, how these cells are regulated in vivo during autoimmune disease settings needs further investigation.MS is an extremely complex autoimmune disease caused by various cellular and molecular mechanisms. From the animal models of MS, it became evident that distinct T helper cell subsets, such as IFN-γ-producing TH17 cells were enriched in the intestine of B10.S mice, but failed to reach the peripheral immune organs, resulting in the absence of spontaneous EAE in B10.S mice. The release of intestine-sequestered TH17 cells by treating B10.S mice with α4β7-specific monoclonal antibodies worsened EAE. We propose that the immune tolerance against myelin-specific self-antigens could be achieved by selective sequestration of pro-inflammatory T cells in the intestine.In this report, we compared the development of spontaneous EAE in myelin-specific TCR transgenic mice on the disease-susceptible SJL/J genetic background with animals on the disease-resistant B10.S background. We found that the pro-inflammatory, myelin-reactive Ts, but on the C57BL/10 background. Although RR SJL/J and RR B10.S mice express the same pair of TCR Vα8.3 and Vβ4 chains on their CD4+ T cells, the incidence of spontaneous EAE was strikingly different. While more than 80% of the RR SJL/J cohort presented spontaneous EAE symptoms between 6–28 weeks of age, none of the B10.S mice displayed any clinical signs of disease (We recently described a transgenic mouse strain that expresses a myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor (TCR) on the SJL/J genetic background (RR mice). The TCR was derived from an encephalitogenic T cell clone generated from recombinant MOG protein (rMOG) immunized wild-type SJL/J mice + T cells in various lymphoid organs of B10.S mice were comparable to those of their SJL/J counterparts (+ regulatory T cells (Treg cells) To determine whether resistance to spontaneous EAE in B10.S animals is due to alterations in T cells, we analyzed T cell populations in the peripheral lymphoid organs. Flow cytometric analysis of the peripheral lymphoid organs showed that the frequencies of CD4s) on B cells from various lymphoid organs confirmed the lower MHC class II expression levels on B10.S APCs compared to APCs from SJL/J mice . Stimulation of the APCs innate immune response by microbial products can result in the upregulation of MHC class II on the surface of B10.S APCs in vivo activation of APCs as well as T cells via immunization with rMOG in CFA did not result in a massive upregulation of the MHC class II-expression on APCs from B10.S mice . Moreover, in the gut associated lymphoid tissues (GALT), where APCs constantly encounter microbial products due to their close proximity to the commensal microbiota, B10.S B cells expressed lower amounts of MHC class II  in B10.S mice. A previous report showed that APCs from B10.S mice express lower amounts of MHC class II We subsequently analyzed if the lower MHC class II-expression on APCs from B10.S mice would result in defective T cell activation and thus, provide an explanation for the absence of spontaneous EAE in RR B10.S mice. However, despite their lower MHC class II-expression, B10.S APCs were capable of processing and presenting the cognate antigen MOG as efficiently to T cells as APCs from SJL/J mice. Moreover, T cells from B10.S mice showed a higher proliferative response to anti-CD3 stimulation than SJL/J T cells, suggesting no intrinsic or extrinsic defect in T cell activation or functional capacity of APCs in B10.S mice  of B10.S mice compared to their SJL/J counterparts  in the intestines of B10.S mice, since colonization levels of SFB were comparable between SJL/J and B10.S animals .Having excluded any general defect in T cell functionality, we sought to characterize the distribution of T helper cell-subsets in B10.S mice. EAE induction in mice has been attributed to distinct T helper cell subsets, which produce either IFN-γ or IL-17, TH17 cells, which are triggered in the intestine. Interestingly, in SJL/J mice, TH17 cells, which were activated in the gut, populate peripheral lymphoid organs and form a pool of pathogenic CD4+ T cells, ready to invade the central nervous system and mediate neurological disease ex vivo analysis of TH17 cells in B10.S mice, however, revealed that despite the higher abundance of TH17 cells in the GALT, frequencies of TH17 cells were greatly reduced in spleen and lymph nodes  responsible for the accumulation of TH17 cells, we treated RR B10.S mice with a blocking antibody to α4β7. Interestingly, blockade of α4β7 led to an enhanced production of IL-17 in the spleen , respectively H17 cells have been shown to be critically regulating the susceptibility to autoimmune diseases H17 cells in the small intestinal lamina propria than SJL/J animals. However, the TH17 cell-inducing load of the segmented filamentous bacteria was similar between SJL/J and B10.S mice. This finding suggests that B10.S mice are indeed capable of mounting a robust TH17 response, but these T cells fail to reach the peripheral lymphoid organs. To investigate whether TH17 cells may have an enhanced migratory affinity towards the intestine of B10.S mice, we transferred TH17 cells into naïve SJL/J and B10.S recipients. Interestingly, we found elevated numbers of TH17 cells in the intestine of B10.S mice compared to SJL/J recipients, suggesting that the B10.S gut harbors a milieu, which is attractive for TH17 cells.While studying immunization models, the effect of the adjuvants on skewing the T helper cell responses cannot be ruled out, we therefore decided to investigate the phenotypes of the T helper cell subsets in a “natural” (unimmunized) state. Striking differences were observed in the frequencies of IL-17-producing T+ dendritic cells H17 cells, resulting in a more severe EAE. Our results suggest that multiple mechanisms are employed to target effector pathogenic TH17 cells to the intestinal mucosa, making them unavailable to participate in pro-inflammatory responses at peripheral sites.It is well appreciated that chemokines and their receptors along with adhesion molecules control the homing pattern of lymphocytes to lymphoid and non-lymphoid tissues. Gut tropism of T cells are mediated by the integrin receptor α4β7 and the chemokine receptor CCR9 that is induced by the gut associated CD103H17 cells in the intestine leading to a reduced frequency of this T helper cell subset in the peripheral repertoire. Understanding the mechanisms that promote the accumulation of pro-inflammatory TH17 cells may help in devising new therapeutic approaches to control MS.In summary, we show here that the resistance to spontaneous EAE in B10.S mice is partly due to enhanced accumulation of activated MOG-specific TA wild-type B10.S mice colony was established from a breeding pair obtained from the McLaughlin Research Institute . TCR transgenic RR SJL/J mice Mice were immunized subcutaneously with 200 µg rMOG emulsified in Freund's adjuvant supplemented with 5 mg/ml Mycobacterium tuberculosis  (CFA). 200 ng of pertussis toxin  were injected intraperitoneally on the day of immunization and 48 hours later. In addition, mice were injected with 250 µg of anti-α4β7 (DATK 32) or isotype control antibodies weekly twice. Clinical signs of EAE were assessed daily according to the standard 5 point scale 3H]thymidine during the final 16 hours of a 72 hour culture period. Results are expressed as mean thymidine uptake (cpm) of triplicate cultures.Splenocytes from RR SJL/J or RR B10.S mice were cultured in the presence of the indicated concentration of rMOG or anti-CD3/anti-CD28 antibodies (BD Pharmingen). The proliferative response was measured by the incorporation of [+ T cells towards TH17 cells was performed as previously described H17 cells were purified by Nycoprep (Axis-Shield). Purified T cells were labelled for 10 min at 37°C with 5 mM CFSE (Life Technologies) in PBS containing 1% fetal bovine serum (FBS). Subsequently, cells were washed twice in ice-cold PBS. 5×106 CFSE-labelled TH17 cells were injected intravenously into wild-type SJL/J or B10.S mice. The frequencies of CFSE+ TH17 cells were measured by flow cytometry after 3 days.Polarization of splenic CD43) with the following fluorochrome-labeled monoclonal antibodies: anti-CD4 (RM4-5), anti-CD3 (145-2C11), anti-B220 (RA3-6B2), anti-I-Aq (KH116), anti-CD86 (GL1), anti-PDL-1 (MIH5), anti-ICOS-L (HK5.3). For intracellular cytokine staining, cells were activated with 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of 5 mg/ml brefeldin A (Sigma) for 4 hours at 37°C. After surface staining, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (eBioscience) and stained intracellularly with the following antibodies: anti-IL17 (TC11-18H10) or anti-FoxP3 (FJK-16s). All antibodies were purchased from BD Pharmingen or eBioscience. Cells were acquired on a FACSCalibur or FACSVerse (BD Biosciences) and analysis was performed using FlowJo (TreeStar) software.Single-cell suspensions were prepared from spleen, pooled peripheral lymph nodes  or Peyer's patches by mechanical disruption via forcing through 40 µm cell strainers (BD Biosciences). Lamina propria lymphocytes were isolated as previously described Cytokine levels in cell culture supernatants were determined using matching antibody pairs for IL-17 (eBioscience) according to manufacturer's instructions.ACTCCTACGGGAGGCAGCAGT and ATTACCGCGGCTGCTGGC; Segmented filamentous bacteria, GACGCTGAGGCATGAGAGCAT and GACGGCACGGATTGTTATTCA.Bacterial genomic DNA was extracted from fecal pellets using QIAamp DNA Stool mini kit (Qiagen). Real-time PCR targeted to 16S rDNA was performed using Absolute QPCR SYBR Green Mix (Thermo Fisher Scientific) and a 7900HT Real-time PCR System (Applied Biosystems). Values were normalized to total bacteria. The following primer sets were used Figure S1Stimulation of the innate immune response does not result in enhanced MHC class II-expression on APCs of B10.S mice. Expression-levels of MHC class II (I-As) and the co-stimulatory/co-inhibitory molecules  on B cells were determined by flow cytometry in the spleen of naïve wild type SJL/J and B10.S mice and wild-type animals 10 days after immunization with rMOG/CFA. Bar graphs show the mean fluorescent intensity (MFI) + SEM on the gated B220+ population. *, p<0.05 (Mann-Whitney U test). Results are from n  = 4–5 mice per group. Data were pooled from 2 independent experiments.(TIF)Click here for additional data file.Figure S2Fecal SFB content in B10.S and SJL/J mice. 16S rDNA PCR for the presence of SFB in the feces of RR SJL/J or B10.S mice. Values are shown as relative amount to total bacterial 16S rDNA.(TIF)Click here for additional data file.


The effector functions of immunoglobulin G (IgG) are mediated by interaction of its Fc region with Fc receptors (FcγRs) and/or the complement system. The three main pathways of complement activation converge at C3. However, C3-independent pathways can activate C5 and other downstream complement components during IgG-initiated inflammatory responses. These C3-independent pathways of C5 activation are triggered by activating FcγRs in some systems or can be activated by factors of the coagulation cascade such as thrombin. Here we studied the interplay of C3, C5, and activating FcγRs in a model of spontaneous autoantibody-driven arthritis.We utilized the K/BxN TCR transgenic mouse model of arthritis. We bred K/BxN mice bearing targeted or naturally-occurring mutations in one or more of the genes encoding complement components C3, C5, and FcRγ, the cytoplasmic signaling chain shared by the activating FcγRs. We measured arthritis development, the production of arthritogenic autoantibodies, T cell activation status and cytokine synthesis. In addition, we treated mice with anti-C5 monoclonal antibodies or with the thrombin inhibitor argatroban.We have previously shown that genetic deficiency of C5 protects K/BxN mice from the development of arthritis. We found here that C3-deficient K/BxN mice developed arthritis equivalent in severity to C3-sufficient animals. Arthritis also developed normally in K/BxN mice lacking both C3 and FcRγ, but could be ameliorated in these animals by treatment with anti-C5 monoclonal antibody or by treatment with argatroban. Production of arthritogenic autoantibodies, T cell activation, and T cell cytokine production were not affected by the absence of C3, C5, and/or FcRγ.In K/BxN mice, C5-dependent autoantibody-driven arthritis can occur in the genetic absence of both complement C3 and activating FcγRs. Our findings suggest that in this setting, thrombin activates C5 to provoke arthritis. The ability of immunoglobulin and immune complexes, including autoantibodies, to provoke inflammation stems from the interaction of the Fc portion of antibody molecules with one or both of two major effector pathways: Fc receptors and the complement system. The relative contributions of these two pathways vary among different disease states and experimental systems -3. A morFcer1g gene) responsible for signal transduction. Mice also express the inhibitory receptor FcγRIIB, whose cytoplasmic tail contains an inhibitory signaling motif. The outcome of an interaction of an FcγR-expressing cell with an IgG-containing immune complex depends on the relative expression levels of the various activating and inhibitory FcγRs and also the IgG subtype (for which the various FcγRs have differing affinities) ..6].in vitro study has recently confirmed that multiple serine proteases in the coagulation and fibrinolysis systems can cleave C3 and C5 to produce C3a and C5a [Several studies have pointed to the existence of additional, C3-independent mechanisms by which C5 can be activated to drive inflammatory responses Figure . More th and C5a . Interpl and C5a ,13-15. HK/BxN T-cell receptor (TCR) transgenic mice spontaneously develop inflammatory arthritis due to combined T- and B-cell recognition of the self-antigen glucose-6-phosphate isomerase (GPI) and production of high-titer anti-GPI IgG autoantibodies ,17. ArthHc allele  [g7 (B6.g7) were also a gift from Mathis and Benoist; I-Ag7 is the mouse major histocompatibility complex (MHC) class II molecule that presents GPI-derived peptides to activate KRN TCR-expressing T cells. C3-deficient mice on the B6 background [Fcer1g)-deficient mice on the B6 background [KRN TCR transgenic mice on the C57BL/6 (B6) background  were a gonal C5) ,23 and Bckground  were a gckground  were purH2) is the only NOD-derived genetic region that the B6.g7 mice retain; to simplify nomenclature, however, we refer to the mice as 'K/BxN' throughout this study as we have previously [C3 and the H2 complex both reside on mouse chromosome 17, a spontaneous chromosomal recombination event was necessary to generate C3-deficient B6.g7 congenic mice. Genotyping of mice was performed by standard polymerase chain reactions. Mice were bred in specific pathogen-free colonies under protocols approved by the University of Minnesota Institutional Animal Care and Use Committee.The C3-, C5-, FcRγ-, and double-deficient 'K/BxN' lines used in this study were created by breeding mice bearing the appropriate knockout allele(s) on the B6 background to KRN/B6 mice and also to B6.g7 congenic mice. The MHC  . AntibodAssessment of arthritis severity by clinical score and ankle thickening, treatment with anti-C5 antibody, determination of anti-GPI IgG titers by enzyme-linked immunosorbent assay, histological analysis, and immunohistochemistry for C3 and IgG were performed as described . For detIntracellular cytokine staining was performed in accordance with the instructions of the manufacturer (eBioscience). Flow cytometry was performed by using a FACSCalibur and an LSRII , and cells were analyzed by using FlowJo V7.6 software .Argatroban  was dissolved in dimethyl sulfoxide (DMSO) and injected intraperitoneally into mice at a dose of 9 mg/kg daily or five days per week fromthe ages of 3 to 6 weeks ,28. No dC3 gene. In agreement with a recent report, we observed that C3-deficient K/BxN mice developed arthritis equivalently to littermate controls  could be at play. Alternatively, antibody fragments with direct prothrombinase catalytic activity have been described .From a clinical perspective, monoclonal antibody reagents designed to interfere with C5 activation systemically or locally  might be effective treatments for inflammatory arthritis Figure  22,38,3,338,39. The key finding of this study is that autoantibody-mediated arthritis in K/BxN mice can occur via a C5 activation pathway that requires neither C3 nor activating FcγRs, the two main effector mechanisms of IgG molecules. Genetic deficiency of C3, C5, and/or FcRγ did not affect T-cell activation or autoantibody production, indicating that the pro-arthritogenic activity of C5 is mediated by its conventional effector mechanisms (likely C5a production). Our data further suggest that thrombin or related proteases of the coagulation cascade mediate C5 activation in the absence of C3 and FcRγ. Understanding how novel pathways of complement activation contribute to autoantibody-mediated arthritis and other inflammatory disorders is expected to lead to new therapeutic approaches.DMSO: dimethyl sulfoxide; FcγR: Fc receptor for immunoglobulin G; FcRγ: the cytoplasmic signaling chain shared by activating Fc receptors for immunoglobulin G; GPI: glucose-6-phosphate isomerase; IFNγ: interferon-gamma; IgG: immunoglobulin G; IL-17: interleukin-17; LPS: lipopolysaccharide; MAC: membrane attack complex; MHC: major histocompatibility complex; NOD: non-obese diabetic; TCR: T-cell receptor; Th17: T helper 17.The authors declare that they have no competing interests.JLA designed and performed experiments, interpreted data, and assisted in writing the manuscript. SSH performed experiments and assisted in writing the manuscript. BAB designed and oversaw experiments, interpreted data, and wrote the manuscript. All authors read and approved the final manuscript.©IgG and C3 deposition in ankles of K/BxN mice lacking C3 or C5 and lacking FcR. Deposition of IgG  and C3  was determined by immunofluorescent microscopy in K/BxN mice expressing or not expressing C3, FcR©, and C5, as indicated in the left column. The key findings are that IgG is deposited in each of the joints. As expected, C3 is absent in the C3-deficient animal . Despite having decreased arthritis severity, C3 is still detectable in the joints of the C5/FcR©-deficient mouse . Slides were counterstained with DAPI (blue) to detect nuclei. Original objective: 40x.Click here for fileHepatic expression of prothrombin/thrombin is upregulated in C3-deficient mice. Liver sections from C3-sufficient (left panels) and C3-deficient (right panels) mice were stained with monoclonal antibodies specific for prothrombin/thrombin (top panels) or isotype control antibodies (bottom panels). Brown staining represents bound antibody. The slides were counterstained with hematoxylin (blue). Original objective: 40x.Click here for file


Cadm1 as a novel modifier of metastasis. We demonstrate that Cadm1 can specifically suppress metastasis without affecting primary tumor growth. Unexpectedly, Cadm1 did not alter tumor-cell-autonomous properties such as proliferation or invasion, but required the host's adaptive immune system to affect metastasis. The metastasis-suppressing effect of Cadm1 was lost in mice lacking T cell–mediated immunity, which was partially phenocopied by depleting CD8+ T cells in immune-competent mice. Our data show a novel function for Cadm1 in suppressing metastasis by sensitizing tumor cells to immune surveillance mechanisms, and this is the first report of a heritable metastasis susceptibility gene engaging tumor non-autonomous factors.Metastasis is a complex process utilizing both tumor-cell-autonomous properties and host-derived factors, including cellular immunity. We have previously shown that germline polymorphisms can modify tumor cell metastatic capabilities through cell-autonomous mechanisms. However, how metastasis susceptibility genes interact with the tumor stroma is incompletely understood. Here, we employ a complex genetic screen to identify  Cadm1 as a metastasis susceptibility gene. Cadm1 was previously identified as a tumor suppressor in lung adenocarcinoma, and reductions in its expression have been associated with poor survival in numerous cancer types. In this manuscript, we use in vivo modeling to show that high expression of Cadm1 inhibits pulmonary metastasis, while knockdown of Cadm1 promotes the metastatic capability of tumor cells. We further show that the metastasis-suppressive effect of Cadm1 expression is lost in mice lacking T cell–mediated immunity and that this effect is partially mediated by CD8+ T-lymphocytes. Our data suggest that the inverse correlation between Cadm1 expression and disease-free survival in humans is a result of a metastasis-suppressive interaction of Cadm1 with the cell-mediated immunity.Metastasis, the dissemination and growth of tumor cells in organs distinct from which they originated, is the most common cause of cancer-related death. Accumulating evidence indicates that an individual's genetic background, the heritable complement of genetic variations that distinguish individuals, not only contributes to overall cancer risk, but also specifically influences metastatic potential. Using a mouse model of metastatic breast cancer and complex genetic analysis, we have identified  Cadm1 as a germline modifier of metastatic susceptibility. We demonstrate that over-expression of Cadm1 by as little as 1.5-fold can specifically suppress metastasis without any resultant difference in primary tumor growth.Metastatic disease remains a major problem for the clinical treatment of many different malignancies. Metastases can appear years after treatment of the primary tumor and is frequently refractory to therapy Cadm1 are lost in mice lacking functional T cell–mediated immunity, an effect which is partially phenocopied by the depletion of CD8+ T cells in immune-competent mice, suggesting that Cadm1 sensitizes tumor cells to immune-surveillance mechanisms by CD8+ T cells. Since differences in expression of Cadm1 are inherited in mice, our data links the contribution of the genetic background in determining metastatic risk to the adaptive immune system, suggesting that individuals with higher levels of Cadm1 expression may be more resistant to metastasis.In addition to tumor-autonomous cellular phenotypes, metastatic efficiency is also impacted by tumor non-autonomous, host-derived factors including the immune system Previous work from our laboratory demonstrated that the progeny of FVB-MMTV-PyMT, a mouse model of metastatic breast cancer, and NZB/B1NJ or C58/J mice have significantly reduced pulmonary metastasis relative to the parental FVB-MMTV-PyMT To further resolve the segment of chromosome 9 carrying this susceptibility locus, three congenic strains were developed that contained overlapping segments of NZB chromosome 9, spanning from 16–67 megabases . Each coCadm1, Zbtb16 and Pias1, had the appropriate haplotype structure and were differentially expressed between the two genotypes. Of the three Cadm1 had the greatest magnitude of differential expression on microarray. Since aberrations in Cadm1 expression have previously been associated with poor prognosis in numerous malignancies Cadm1 was selected for further characterization. Zbtb16 and Pias1 are under investigation in separate studies.To filter the gene list in the chromosome 9 metastasis modifier interval for potential candidate genes, haplotype analysis was performed. Based on the hypothesis that NZB and C58 shared a common metastasis susceptibility gene, screening for genes with identical haplotypes in NZB and C58 that were distinct from the FVB haplotype identified 80 potential candidate genes. Since the majority of polymorphisms occur in non-coding sequence it is hypothesized that the majority of quantitative traits are due to subtle changes in gene expression rather than non-synonymous amino acid substitutions. Based on this assumption the candidate gene list was further filtered by RNA expression microarray analysis. Tumors from PyMT-FVB or NZB chromosome 9 substitution (NF9) strains were subjected to microarray analysis to identify differentially expressed transcripts. Three genes within the candidate interval, Cadm1 protein-coding exons was performed to confirm the predicted haplotype differences between NZB and FVB and to screen for undescribed polymorphisms. Sequence analysis confirmed that NZB and FVB have distinct haplotypes at the Cadm1 locus and validated one synonymous polymorphism in exon 2  was conducted on reverse transcribed RNA extracted from PyMT-FVB and NF9 tumors. The results showed a trend toward increased Cadm1 expression in NF9 tumors relative to FVB that was of borderline significance , a marker of activated CD8+ T cells and NK-cells + T cells, enhance CD8+ T cell and NK-cell cytotoxicity in vitro, and tumor rejection in vivoCadm1-mediated metastasis suppression, control and Cadm1-expressing cells were orthotopically implanted into athymic nude mice. As illustrated in Cadm1-expressing cells was rescued in immunocompromised mice. In addition, the tumor suppressive effect of Cadm1 in Mvt-1 cells was also lost. This suggests that T cell mediated immunity may be critical for the tumor and metastasis suppressive effects of Cadm1.Crtam is present on activated natural killer (NK) and CD8+ T cells. Since athymic mice possess functional NK cells, we hypothesized that the loss of Cadm1-mediated metastasis suppression might be mediated by CD8+ T cells. Thus we orthotopically injected Mvt-1 cells expressing Cadm1 in immune-competent mice in which CD8+ lymphocytes were depleted with anti-CD8 antibody , a web-based meta-analysis tool containing microarray-based tumor expression data on 1881 patients from 11 public datasets The down-regulation of Cadm1 expression consistently correlated with significantly improved survival in estrogen receptor (ER) positive tumor datasets,  knockdown of endogenous Cadm1 potentiates metastatic capability, and 2) pulmonary metastases have lost expression of exogenous Cadm1. Together with the numerous Cadm1 expression is lost in more invasive tumors, our data implicates Cadm1 loss as an important step in cancer immunoediting that enhances the immune evasive and metastatic properties of tumor cells.Further, the partial rescue of the metastatic phenotype by depletion of CD8The NZB backcross has been described previously The C58 backcross was generated by crossing PyMT male animals with C58/J females to generate PyMT-positive F1 males, which were subsequently bred to FVB/NJ females to generate N2 backcross animals. Tumor phenotyping was performed as previously described Exons were amplified from genomic DNA then run on a 2% agarose gel. Amplified bands were purified by the Qiagen Gel Extraction Kit and cloned into TOPO-TA vectors (Invitrogen) and transformed into One-shot Top10 chemically competent E. Coli (Invitrogen) as per manufacture's protocol. Plasmid was purified using the Mini Prep kit (Qiagen). Sequencing reactions were conducted on ∼350 ng of purified plasmid using Big Dye Terminator reaction mix (Applied Biosystems) following the manufacturers protocol and sequenced by the NCI Sequencing Facility.All experiments were conducted using FVB/NJ background mouse mammary tumor explants cell lines 6DT1 and Mvt-1 kindly provided by Dr. Lalage Wakefield 5′-GGGGACAACTTTGTACAAAAAAGTTGGCACCATGGCGAGTGCTGTGCTGCCGAGCGGATCpDonr253 is a Gateway Donor vector modified from pDonr201 (Invitrogen). pDonr253 replaces the kanamycin resistance gene with a gene encoding spectinomycin resistance, and contains several sequencing primer sites to aid in sequence verification of Entry clones. The following oligonucleotides (Eurofins MWG Operon) were used in this study: L10379: 5′-GGGGACAACTTTGTACAAGAAAGTTGAGATGAAGTACTCTTTCTTTTCTTCL10383: Cadm1 knockdown wereThe sequences of short hairpin RNA pLKO.1 vectors for CCGGCGGACTGGTTTGTAAAGGAAACTCGAGTTTCCTTTACAAACCAGTCCGTTTTTGshRNA14: CCGGCCTGTTCATCAATAACCTAAACTCGAGTTTAGGTTATTGATGAACAGGTTTTTGshRNA15: Cadm1 was cloned into a Gateway Entry clone by PCR from a cDNA template . The Entry clone contains the complete ORF preceded by a Kozak translation initiation sequence and an ATG start codon, and lacking a stop codon at the 3′ end to allow C-terminal fusions to be generated. DNA was amplified using specific primers L10379 and 10383 containing the Gateway recombination sequences, and PCR was carried out using Pfusion polymerase (New England Biolabs). The final PCR product contains the gene of interest flanked on the 5′ side with a Gateway attB1 site and on the 3′ side with a Gateway attB2 site. The PCR product was cleaned using the QiaQuick PCR purification kit (Qiagen), and recombined into pDonr253 using the Gateway BP recombination reaction (Invitrogen) by the manufacturer's protocols. The subsequent Entry clone was sequence verified throughout the entire cloned region.Murine Cadm1 was generated using Multisite Gateway recombination. An entry clone using the murine Pol2 promoter was recombined with the Cadm1 entry clone and a C-terminal entry clone encoding the V5 epitope tag (GKPIPNPLLGLDST) into a Gateway destination vector pDest-659. This vector is a modified version of the pFUGW lentiviral vector which contains the enhanced polypurine tract (PPT) and woodchuck regulatory element (WRE) to provide higher titer virus. In addition, it contains an antibiotic resistance gene for blasticidin resistance. Entry clones were subcloned by Gateway Multisite LR recombination using the manufacturer's protocols (Invitrogen). Expression clones were transformed into E. coli STBL3 cells to minimize unwanted LTR repeat recombination, and verified by agarose gel electrophoresis and restriction digest. Transfection-ready DNA for the final clones was prepared using the GenElute XP Maxiprep kit (Sigma). A control vector (8166-M24-658) was generated by standard Gateway LR recombination of a stuffer fragment made up of a non-coding DNA into the pLenti6-V5-DEST vector (Invitrogen). All lentivector constructs and lentivirus particles were generated by the Protein Expression Laboratory and the Viral Technology Group in NCI, Frederick.A lentiviral vector expressing a C-terminal V5 epitope-tagged fusion of 4 cells were incubated at 37°C in 5% CO2 overnight. Cells were then infected with 50 uL of concentrated lentivirus suspension, and selected 30 hours post-infection with 5 mg/mL blasticidin for over-expression (Invitrogen) constructs or 10 ug/mL (shRNA) puromycin for shRNA constructs.Two milliliter suspensions of 5×10Protein was extracted by cell lysis in 400 uL of Pierce lysis buffer, vigorous homogenization, and incubation on ice for one hour. Twenty micrograms of protein extract per sample in NuPage LDS Sample Buffer and NuPage Reducing Agent (Invitrogen) were used for western blotting. PVDF membrane (Millipore) containing transferred proteins was incubated overnight with the primary antibodies: mouse anti-V5 (Invitrogen), chicken IgY anti-SynCAM/TSLC1/Cadm1 , or mouse anti-β-actin (Abcam). The membrane was then incubated with horse-radish peroxidase linked anti-mouse  or anti-chicken IgY secondary antibodies (Abcam). Immunoblot was visualized using Amersham ECL Prime Western Blotting Detection System and Amersham Hyperfilm ECL . Densitometry data were obtained and analyzed with a ChemiDoc-It Imaging System and VisionWorksLS software (UVP).RNA was isolated from tumors and cell lines using RNeasy kit (Qiagen) and reverse transcribed using iScript (Bio-Rad). Real-Time PCR was conducted using QuantiTect SYBR Green PCR kit (Qiagen). See 5 cells were seeded in triplicate into each well of 24-well Essen ImageLock (Essen Bioscience) plates in selective media and incubated at 37°C and 5% CO2 overnight. The following day, the cells washed twice with PBS and incubated at 37°C and 5% CO2 for 3 hours in 10 ug/mL mitomycin C. Scratch wounds were made using Essen 4-channel scratch instrument loaded with Eppendorf 10 uL micropipette tips. Cells were subsequently washed three times with PBS, placed in selective media and placed in Incucyte (Essen Bioscience). Incucyte was programmed to image each well at 2-hour intervals. Data analysis was conducted using Incucyte 2011A software.One-milliliter suspensions of 6×103 cells were seeded in six replicates into each well of 24-well cell culture plates  in selective media and placed in Incucyte. Incucyte was programmed to image each well at 3-hour intervals. Data analysis was conducted using Incucyte 2011A software.One-milliliter suspensions of 5×104 cells were seeded in triplicate into each well of the 24-well plate format BD BioCoat Control and Matrigel Invasion Chambers (BD Biosciences) and incubated for 20 hours at 37°C in 5% CO2. Enriched media was used as chemoattractant. Membranes were subsequently fixed with methanol, stained with crystal violet, and cells present on the underside of the membrane were counted.Matrigel inserts were hydrated for 2 hours in 500 uL serum-free DMEM then suspensions of 7.5×102 for 3 days prior to microscopy.Cultrex 3-D culture matrix  was injected into 8-chamber slides and allowed to solidify at 37°C for 30 minutes. Cells were trypsinized and 5,000 counted then resuspended in 400 uL DMEM, 9%FBS, 5 ug/mLBlasticidin, and 2% Cultrex and incubated at 37°C with 5% CO5 cells were injected orthotopically into the fourth mammary fat pad of age-matched virgin females. After 30 days the mice were euthanized by intraperitoneal injection of 1 mL Tribromoethanol with subsequent cervical dislocation. Primary tumors were resected, weighed, and snap frozen in liquid nitrogen. Lungs were resected, surface metastases were counted; lungs were inflated with 10% nitrate-buffered formalin and sent for sectioning and staining. For tail vein injection, 7×105 were injected into the lateral tail vein, mice were euthanized 22 days post-injection. For the CD8-antibody treatment study, orthotopic transplantation was conducted but mice were treated with 0.5 mg of either control rat IgG or rat monoclonal anti-CD8 IgG  on days −4, −3, −2, +3, +10, +17, +24, and +30; cells were injected on day 0 and mice were euthanized on day 37. All procedures were performed under the Animal Safety Proposal (LCBG-004) and approved by the NCI-Bethesda Animal Care and Use Committee.Female FVB/NJ mice from Jackson Laboratories were injected at 6–8 weeks of age. Two days prior to orthotopic injections, cells were placed in non-selective media. On the day of injection, 1×10Immunohistochemical staining was performed on LeicaBiosystems' Bond Autostainer on paraffin embedded tissue sections with biotinylated rabbit anti-Goat IgG; primary antibody, anti-V5 rabbit polyclonal antibody ; LeicaBiosystems Intense R Detection Kit. All histological analysis including paraffin embedding, sectioning, hematoxylin and eosin staining and immunohistochemistry were conducted by the Pathology/Histotechnology Laboratory, Laboratory Animal Sciences Program, SAIC, Frederick.Cadm1-expressing or control tumor bearing mice: Single cell suspensions from the lymph nodes of tumor bearing mice were prepared. 1.6 million cells were loaded into each well of an IFN-γ ELISPOT plate, stimulated with anti-CD3 , and cultured overnight. The procedure was done according to recommendations from the manufacture (BD). Three to four mice for each experimental group, and triplicates for each sample were examined. The ELISPOT plate was scanned in ImmunoSpot  and quantification was assessed using the CTL Scanning and CTL counting 4.0.For IFN-γ production by T cells in either Statistical analysis comparing two samples were conducted using the Mann-Whitney test on Prism Version 5.03 . Multiple-comparison data was analyzed by Kruskal-Wallis test with post-hoc Conover-Inman correction for multiple analyses by R-script. Survival data was conducted with the Mantel-Cox test on Prism.Figure S1NZB and C58 chromosome 9 metastasis QTL.(EPS)Click here for additional data file.Figure S2Cadm1 exon 2 (rs32721609).Detection of synonymous SNP in (EPS)Click here for additional data file.Figure S32) was measured on hematoxylin and eosin stained sections, no statistically significant difference was observed .Metastatic nodule cross-sectional area (um(EPS)Click here for additional data file.Figure S4Cadm1 expressing Mvt-1 and 6DT1 cells relative to control.No significant difference observed in the growth rate or cellular phenotype of (TIF)Click here for additional data file.Figure S5Growth in three-dimensional culture. After growth Cultrex-containing rich media for 3 days, cells showed no difference in growth in three dimensional culture.(EPS)Click here for additional data file.Figure S6Cadm1 expression on trans-well migration and invasion. Cadm1-expressing Mvt-1 (a and c) and 6DT1 (b and d) cells were assayed for serum-stimulated transwell migration through porous Polytetrafluoroethylene and invasion through matrigel.Effect of (EPS)Click here for additional data file.Figure S7Cadm1 expressing Mvt-1 and 6DT1 cells relative to control. Cadm1 expression had no effect on Mvt-1 cell motility but resulted in a minor reduction in motility of 6DT1 cells.Motility of (TIF)Click here for additional data file.Figure S8+ T cell depletion in the peripheral blood of one representative sample from each group .Flow cytometry demonstrating CD8(EPS)Click here for additional data file.Figure S9Cadm1 expression in Mvt-1 cell lines directly prior to injection into animals for in vivo studies in immune-competent (FVB), athymic (nude), and antibody treated mice.Protein-level (EPS)Click here for additional data file.Table S1Breast cancer patient dataset details. n, number of patients per group; LN, lymph node; ER, estrogen receptor; IDC, invasive ductal carcinoma; TCGA, The Cancer Genome Atlas.(EPS)Click here for additional data file.Table S2Primer sequences.(DOC)Click here for additional data file.