High-throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire, which makes it difficult to correlate T cell reconstitution with clinical outcomes. The associations identified thus far are of a broadly statistical nature, precluding precise modeling of outcomes based on T cell repertoire development following bone marrow transplantation (BMT). Previous work has demonstrated an inherent, mathematically definable order observed in the T cells from a diverse group of donors, which is perturbed in recipients following BMT. In this study, T cell receptor (TCR)-b sequences from HLA-matched related donor and recipient pairs are analyzed to further develop this methodology. TCR-b sequencing from unsorted and sorted T cell subsets isolated from the peripheral blood samples of BMT donors and recipients show conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR-b VJ segment translational symmetry is preserved post-transplantation and even in cases of acute graft-versus-host disease (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antigens. The complexity of the repertoire is significantly diminished after BMT, and the T cell clonal hierarchy is altered post-transplantation. Low-frequency donor clones tended to take on a higher rank in the recipients following BMT, especially in patients with aGVHD. Over time, the repertoire evolves to a more donor-like state in the recipients who did not develop GVHD as opposed to those who did. The results presented here support new methods of quantifying and characterizing post-transplantation T cell repertoire reconstitution.

Background Major histocompatibility complex (MHC) class-1 antigen is a glycoprotein expressed in all nucleated cells. The aim of this study was to assess MHC class-I expression in different neuromuscular diseases. Methods The authors reviewed the data of 54 patients with neuromuscular diseases. Anti MHC class-I antibody was performed on the frozen muscle tissues using immunohistochemistry. MHC class-I was scored based on its expression on muscle fibers (0: normal, 1: expression <5 fibers, 2: expression in 5–10 fibers, 3: expression in >10 fibers). The pattern was only assessed in cases with MHC class-I scored 3 as: (1: Sarcocapillary, 2: Sarcocapillary and necrotic fibers, 3: Perifascicular). The relationship between MHC class-I expression and neuromuscular diseases was statistically analyzed. Results The mean age of the patients was 39.1 ± 18.5 years. Around 50% of patients showed normal CK levels and 5% of the cases showed elevated CK levels. There was a significance difference in MHC class-I expression between cases with normal and elevated CK levels when MHC class-I score was 3 (p= 0.020). There was a significant difference in MHC class-I expression among different neuromuscular diseases (p<0.001). All cases with idiopathic inflammatory myopathies (IIMs) have expressed MHC class-I in more than 10 fibers. MHC class-I was expressed in 15 cases of non-IIMs. Conclusion MHC class-I cannot be solely used as a biomarker to distinguish IIMs from non-IIMs. The presence of MHC class-I molecules in non-IIMs might be related to immunoproteasomes mechanism. Further studies, with different muscle proteins expression and genomic sequencing, must be conducted to understand the role of MHC Class-I in neuromuscular diseases.

281 words; Manuscript 7895 words; Figures 6; Tables 2; Supplementary figures 7; Supplementary table 1. Abstract High throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire. In an attempt to correlate T cell reconstitution with clinical outcomes several measures of T cell repertoire complexity have emerged. However, the associations identified are of a broadly statistical nature, not allowing precise modeling of outcomes based on T cell repertoire development in clinical contexts such as following bone marrow transplantation (BMT). Previous work demonstrated that there is an inherent, mathematically definable order observed in the T cell population that is conserved in a diverse group of donors, and which is perturbed in recipients following BMT. Herein, we use a public database of human leukocyte antigen matched related-donor and recipient T cell receptor (TCR) b sequences to further develop this methodology. TCR b sequencing from unsorted T cells and sorted T cell subsets isolated from peripheral blood samples from BMT donors and recipients show remarkable conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR b VJ segment translational symmetry is preserved post-transplant, and even in cases of acute GVHD (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antiges. We also observe that the complexity of the repertoire is significantly diminished after BMT and is not restored even years out posttransplant. The results here provide a new method of quantifying and characterizing post-transplant T cell repertoire reconstitution by further analyzing the mathematical rules governing TCR usage in the context of BMT. This approach may allow for a new means to correlate clinical outcomes with the evolving T cell repertoire post-transplant.

expanded cord blood-as a means to overcome immunodeficiency after HSCT. To assess this, CD34+ UCB cells were first expanded in the presence of SR-1 and cytokines for 15 days; a portion of SR-1 expanded cells were then co-cultured with murine OP9-DL1 cells with cytokines for an additional 14 days. We found that SR-1 expanded cord blood used as input for Tprog culture resulted in hundreds of millions of Tprogs due the in large input of cells. We also compared the phenotypes of SR-1 and non-expanded (control) Tprogs throughout the 14-day culture. Interestingly, the phenotype of the SR-1 UCB Tprogs was very different than control Tprogs in regards to CD34 expression. SR-1 UCB Tprogs had significantly less CD34+CD7+ cells compared to control UCB (5% and 25%, respectively, P < .01). To assess immune reconstitution in vivo of SR-1 expanded UCB Tprogs, we utilized a humanized mouse model with neonatal nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γc null mice (NSG mice). Neonatal mice were injected with 1e6 SR-1 Tprogs that had been sorted as CD34 + CD7 + or CD34 − CD7 + subsets, and thymuses were assessed after 4 weeks. Both control and SR-1 CD34 + CD7 + Tprogs showed robust thymic engraftment; however, SR-1 Tprogs CD34 − CD7 + cells were uniquely able to engraft the thymus in mice. The thymic cellularity was~60% lower in mice engrafted with CD34 − CD7 + compared to CD34 + CD7 + SR-1 Tprogs (Figure 1, P < .05). Taken together, these data support a novel methodology for generating scalable numbers of Tprogs capable of thymic engraftment that can be translated to patients undergoing HSCT as a potential approach to decrease infections and mitigate relapse risk.