Citations

Abstract The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on recent thymic emigrants, and leaving very few CD4+T cells in peripheral lymphoid organs. Interestingly, thymic- and splenic-derived T cells from CD4-Cre Runx1 cKO mice exhibit increased expression of markers indicative of anergy including FR4 and CD73, which is not seen in other genetically modified mice with a block in T cell maturation. These data suggest Runx1 plays a role in regulating the development of T cell anergy in CD4+T cells. We have generated a 1:1 mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre (ER-Cre) Runx1 conditional knockout and wild type B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, the ER-Cre Runx1 cKO CD4+ T cells spontaneously develop an anergic phenotype as they fail to proliferate upon TCR/CD28 stimulation, and co-express CD73 and FR4. This cell surface phenotype is cell intrinsic since wild type B6.SJL CD4+ T cells in the mixed bone marrow chimera do not upregulate CD73 and FR4 upon tamoxifen treatment. Surprisingly, the mixed BMC system reveals both the ER-Cre Runx1 cKO and the wild type B6.SJL peripheral T cells fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 also regulates a cell-extrinsic suppressive mechanism controlling T cell tolerance. This cell-extrinsic induction of anergy requires Runx1-deficient CD4+ T cells, but it is not dependent on Runx1-deficient Tregs. Thus, Runx1 is a key regulator of T cell anergy, and further analysis of this BMC model will serve to uncover the mechanism.

Abstract The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on CD4+ recent thymic emigrants, leaving very few CD4+ T cells in peripheral lymphoid organs. Consistent with a block in maturation, peripheral Runx1-deficient CD4+ T cells fail to produce normal levels of TNF-a upon stimulation. These CD4+ T cells that persist in CD4-Cre Runx1 cKO mice possess a phenotype not found in other genetically modified mice with a block in T cell maturation. Thymic- and splenic-derived T cells from the CD4-Cre Runx1 cKO exhibit altered expression of markers indicative of anergy including FR4 and CD73. This suggests Runx1 plays a role in regulating the development of T cell tolerance which is disrupted upon deletion of Runx1 in CD4+ T Cells. To test this, we have generated a mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre Runx1 knockout and B6.SJL bone marrow to knock out Runx1 in mature T cells. Surprisingly, in the mixed BMC both the ER-Cre Runx1 cKO and the B6.SJL peripheral T cells acquire a functionally hyporesponsive phenotype, suggesting that Runx1 regulates an extrinsic signaling mechanism controlling tolerance. Moving forward, this BMC model allows us to investigate the development of this anergic phenotype in mature T cells, and tests the hypothesis that Runx1 plays a critical role in regulating T cell tolerance.

Abstract The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.

Abstract Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.

Abstract Spleen cells from a number of H-2 congenic mice were tested for their proliferative and/or cytolytic responses to partially purified products from CNBr cleavage of Sendai virus. In response to in vitro stimulation of Sendai-primed spleen cells with virus-infected cells, Sendai virus-specific cytolytic T lymphocytes restricted to H-2Kb, Kd, Kk, Kb, and Dd were generated. Of these, H-2Kb- and Kd-, but not H-2Kk-, Kb-, and Dd-, restricted cytolytic T cells were generated after stimulation with partially purified Sendai peptides. H-2Dd-restricted, virus-specific cytotoxicity was generated in response to unseparated products from CNBr cleavage of Sendai virus. Thus, the pool of partially purified peptides contains some but not all of the antigenic determinants that can stimulate a cytolytic response. These results suggest that each determinant on Sendai viral proteins may stimulate cytolytic T lymphocytes restricted to only a subset of the H-2K and D alleles which can be recognized by the sum of cytolytic T cells generated in response to all of the antigenic determinants on Sendai viral proteins. Thus, H-2K and D genes function as immune response genes regulating the cytolytic response to partially purified peptides and, by extension, possibly to individual antigenic determinants on Sendai viral proteins. Also, spleen cells from three mice differing only in the H-2I-S regions show differences in their proliferative responses to Sendai antigens. Only spleen cells from mice with the k haplotype in IA-IJ proliferate in response to partially purified peptides, whereas spleen cells from all three mice proliferate when virus is added to the cultures. These proliferating cells are sensitive to treatment with anti-Thy-1.2 and C, suggesting that immune response genes regulating T cell proliferative responses to individual viral determinants may also exist.

Lynch syndrome (LS), caused by heterozygous pathogenic variants affecting one of the mismatch repair (MMR) genes (MSH2, MLH1, MSH6, PMS2), confers moderate to high risks for colorectal, endometrial, and other cancers. We describe a four-generation, 13-branched pedigree in which multiple LS branches carry the MSH2 pathogenic variant c.2006G>T (p.Gly669Val), one branch has this and an additional novel MSH6 variant c.3936_4001+8dup (intronic), and other non-LS branches carry variants within other cancer-relevant genes (NBN, MC1R, PTPRJ). Both MSH2 c.2006G>T and MSH6 c.3936_4001+8dup caused aberrant RNA splicing in carriers, including out-of-frame exon-skipping, providing functional evidence of their pathogenicity. MSH2 and MSH6 are co-located on Chr2p21, but the two variants segregated independently (mapped in trans) within the digenic branch, with carriers of either or both variants. Thus, MSH2 c.2006G>T and MSH6 c.3936_4001+8dup independently confer LS with differing cancer risks among family members in the same branch. Carriers of both variants have near 100% risk of transmitting either one to offspring. Nevertheless, a female carrier of both variants did not transmit either to one son, due to a germline recombination within the intervening region. Genetic diagnosis, risk stratification, and counseling for cancer and inheritance were highly individualized in this family. The finding of multiple cancer-associated variants in this pedigree illustrates a need to consider offering multicancer gene panel testing, as opposed to targeted cascade testing, as additional cancer variants may be uncovered in relatives.

Abstract1q gain (+1q) is the most common high‐risk cytogenetic abnormality (HRCA) in patients with multiple myeloma (MM). However, its prognostic value remains unclear in the era of novel agents. Here, we retrospectively analyzed the impact of +1q on the outcomes of 934 patients newly diagnosed with MM. +1q was identified in 53.1% of patients and verified as an independent variate for inferior overall survival (OS) (hazard ratio, 1.400; 95% confidence interval, 1.097–1.787; p = .007). Concurrence of other HRCAs (particularly t(14;16) and del(17p)) further exacerbated the outcomes of patients with +1q, suggesting prognostic heterogeneity. Thus, a risk‐scoring algorithm based on four risk variates (t(14;16), hypercalcemia, ISS III, and high LDH) was developed to estimate the outcomes of patients with +1q. Of the patients, 376 evaluable patients with +1q were re‐stratified into low (31.6%), intermediate (61.7%), and high risk (6.7%) groups, with significantly different progression‐free survival and OS (p < .0001), in association with early relapse of the disease. The prognostic value of this model was validated in the CoMMpass cohort. While attaining undetectable MRD largely circumvented the adverse impact of +1q, it scarcely ameliorated the outcome of the patients with high risk, who likely represent a subset of patients with extremely poor survival. Hence, patients with +1q are a heterogeneous group of high‐risk patients, therefore underlining the necessity for their re‐stratification. The proposed simple risk‐scoring model can estimate the outcomes of patients with +1q, which may help guide risk‐adapted treatment for such patients.

AbstractBackgroundCurrent CT quality control (QC) for low‐contrast detectability relies on visual inspection and measurement of contrast‐to‐noise ratio (CNR). However, CNR numbers become unreliable when it comes to nonlinear methods, such as iterative reconstruction (IR) and deep‐learning‐based techniques. Image quality metrics using channelized Hotelling observer (CHO) have been validated to be well correlated with human observer performance on phantom‐based and patient‐based tasks, but it has not been widely used in routine CT QC mainly because the CHO calculation typically requires a large number of repeated scans in order to provide accurate and precise estimate of index of detectability (d′).PurposeThe main goal of this work is to optimize channel filters and other CHO parameters and accurately estimate the low‐contrast detectability with minimum number of repeated scans for the widely used American College of Radiology (ACR) CT accreditation phantom so that it can become practically feasible for routine CT QC tests.MethodsTo provide a converged d′ value, an ACR phantom was repeatedly scanned 100 times at three dose levels (24, 12, and 6 mGy). Images were reconstructed with two kernels (FBP Br44 and IR Br44‐3). d′ as a function of number of repeated scans was determined for different number of background regions of interest (ROIs), different number of low‐contrast objects, different number of slices per each object, and different channel filter options. A reference d′ was established using the optimized CHO setting, and the bias of d′ was quantified using the d′ calculated from all 100 repeated scans. The variation of d′ at each condition was estimated using a resampling method combining random subsampling among 100 repeated scans and bootstrapping of the ensembles of signal and background ROIs.ResultsOptimized parameters in CHO calculation were determined: two background ROIs per object, four objects per low‐contrast object size, nine non‐overlapping slices per object, and a 4‐channel Gabor filter. The bias and uncertainty were estimated at different numbers of repeated scans using these parameters. When only one single scan was used in the CHO calculation, the bias of d′ was below 6.2% and the uncertainty 15.6‐19.6% for the 6, 5, and 4 mm objects, while with three repeated scans the bias was below 2.0% and uncertainty 8.7‐10.9% for the three object sizes.ConclusionWith optimized parameter settings in CHO, efficient and accurate measurement of low‐contrast detectability on the commonly used ACR phantom becomes feasible, which could potentially lead to adoption of CHO‐based low‐contrast evaluation in routine QC tests.

AbstractHow to deliver best care in various clinical settings remains a vexing problem. All pertinent healthcare-related questions have not, cannot, and will not be addressable with costly time- and resource-consuming controlled clinical trials. At present, evidence-based guidelines can address only a small fraction of the types of care that clinicians deliver. Furthermore, underserved areas rarely can access state-of-the-art evidence-based guidelines in real-time, and often lack the wherewithal to implement advanced guidelines. Care providers in such settings frequently do not have sufficient training to undertake advanced guideline implementation. Nevertheless, in advanced modern healthcare delivery environments, use of eActions (validated clinical decision support systems) could help overcome the cognitive limitations of overburdened clinicians. Widespread use of eActions will require surmounting current healthcare technical and cultural barriers and installing clinical evidence/data curation systems. The authors expect that increased numbers of evidence-based guidelines will result from future comparative effectiveness clinical research carried out during routine healthcare delivery within learning healthcare systems.