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Colorectal cancer is the second leading cause of cancer death worldwide, and the incidence of this disease is expected to increase as global socioeconomic changes occur. Immune checkpoint inhibition therapy is effective in treating a minority of colorectal cancer tumors; however, microsatellite stable tumors do not respond well to this treatment. Emerging cancer immunotherapeutic strategies aim to activate a cytotoxic T cell response against tumor-specific antigens, presented exclusively at the cell surface of cancer cells. These antigens are rare and are most effectively identified with a mass spectrometry-based approach, which allows the direct sampling and sequencing of these peptides. Although the few tumor-specific antigens identified to date are derived from coding regions of the genome, recent findings indicate that a large proportion of tumor-specific antigens originate from allegedly noncoding regions. Here, we employed a novel proteogenomic approach to identify tumor antigens in a collection of colorectal cancer-derived cell lines and biopsy samples consisting of matched tumor and normal adjacent tissue. The generation of personalized cancer databases paired with mass spectrometry analyses permitted the identification of more than 30,000 unique MHC I-associated peptides. We identified 19 tumor-specific antigens in both microsatellite stable and unstable tumors, over two-thirds of which were derived from noncoding regions. Many of these peptides were derived from source genes known to be involved in colorectal cancer progression, suggesting that antigens from these genes could have therapeutic potential in a wide range of tumors. These findings could benefit the development of T cell-based vaccines, in which T cells are primed against these antigens to target and eradicate tumors. Such a vaccine could be used in tandem with existing immune checkpoint inhibition therapies, to bridge the gap in treatment efficacy across subtypes of colorectal cancer with varying prognoses. Data are available via ProteomeXchange with identifier PXD028309.

Summary The PSMB11 proteasomal subunit, expressed only in cortical thymic epithelial cells (cTECs), is essential for the development of functional CD8+ T cells. An attractive yet unproven theory holds that PSMB11 generates unique major histocompatibility complex class I (MHC I)-associated peptides required for positive selection. We recently reported that PSMB11 regulates the expression of hundreds of genes in cTECs, mainly by differential proteolysis of transcription factors. Thereby, PSMB11 maintains the distinctness of cTECs relative to medullary TECs (mTECs) and promotes cortex-to-medulla migration of developing thymocytes. These conclusions have been challenged by Ohigashi and colleagues, who suggest that their data show that PSMB11 uniquely controls antigen presentation without affecting cTEC biology. Here, we perform a comprehensive reanalysis of transcriptomic and proteomic data from the Ohigashi lab and confirm our original conclusions. This Matters Arising paper is in response to Ohigashi et al. (2019) , published in Cell Reports. See also the response by Ohigashi and Takahama (2021) , published in this issue of Cell Reports.

Introduction Rapid T cell reconstitution following hematopoietic stem cell transplantation is essential for protection against infections and has been associated with lower incidence of chronic graft-vs-host disease (cGVHD), relapse and transplant-related mortality (TRM). While cord blood (CB) transplants are associated with lower rates of cGVHD and relapse, their low stem cell content results in slower immune reconstitution and higher risk of graft failure, severe infections and TRM. Recently, results of a Phase I/II trial revealed that single UM171-expanded CB transplant allowed the use of smaller CB units without compromising engraftment. We now report on T cell reconstitution and immune function in patients transplanted with UM171-expanded CB grafts. Methods We performed a retrospective analysis of 20 patients treated with UM171-expanded CB and compared it to a contemporary cohort of 12 patients treated in the same institution who received unmanipulated CB transplant with similar conditioning regimens. Of note, no patient received ATG as part of the conditioning in either cohort. We used flow cytometry and TCR sequencing to evaluate T cell reconstitution, and virus-specific ELISpot assays to evaluate T cell function in the first year post-transplantation. We also categorized infectious events as per definitions of infection severity in the BMT CTN Technical MOP Version 3.0 and report the mean cumulative count of infectious events for each cohort. Results While median T cell dose in graft was at least 2-3x lower for the cohort of patients treated with UM171-expanded CB due to the selection of smaller cords and to cell loss occurring during CD34 selection process, numbers and phenotype of T cells at 3, 6 and 12 months post-transplant were similar in patients treated with UM171-expanded or unmanipulated CB transplant. TCR sequencing analyses revealed that UM171 patients had greater T cell diversity and higher numbers of T cell clonotypes at 12 months post-transplant compared to patients who received unmanipulated CB. Younger UM171 patients (i.e. Conclusion Our data show that the relative T-cell paucity of the UM171 graft is rapidly compensated after transplant with no significant difference observed between the two cohorts in terms of numbers and phenotypes of T cells at 3, 6 or 12 months post-transplant. Although it is difficult to dissect the relative contribution of homeostatic expansion and de novo thymopoiesis, recipients of UM171 grafts had a greater TCR diversity at one year, which was more evident among patients younger than 40 years of age. The prompt immune reconstitution observed in UM171 patients translated into a low rate of severe (grade 2-3) infections and no infection-related mortality. These results support rapid and functional T cell reconstitution following UM171 expanded CB transplantation, which likely contributes to the absence of moderate/severe cGVHD, infection-related mortality and late TRM observed in this cohort. Figure legend: Mean cumulative counts of infectious events in patients transplanted with UM171-expanded (blue) or unmanipulated (red) CB. Mean cumulative counts are shown for all infectious events (A), bacterial (B) and viral (C) infections. Events were categorized by type and severity as per BMT CTN guidelines (Appendix 4A). Infectious events of grade 1-3 are shown in pale colors, while more severe events (grade 2-3) are shown in dark colors. Censored patients (including those who relapsed) are indicated with white circles. Figure 1 Disclosures Dumont-Lagacé: ExCellThera: Current Employment. Busque:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria. Sauvageau:ExCellThera: Current equity holder in private company, Other: CEO, Patents & Royalties. Cohen:ExCellThera: Consultancy, Other: principal investigator of an ongoing UM171 clinical trial.

T-cell development and function are regulated by MHC-associated self peptides, collectively referred to as the immunopeptidome. Large-scale mass spectrometry studies have highlighted three key features of the immunopeptidome. First, it is not a mirror of the proteome or the transcriptome, and its content cannot be predicted with currently available bioinformatic tools. Second, the immunopeptidome is more plastic than previously anticipated, and is molded by several cell-intrinsic and cell-extrinsic factors. Finally, the complexity of the immunopeptidome goes beyond the 20-amino acids alphabet encoded in the germline, and is not restricted to canonical reading frames. The large amounts of 'dark matter' in the immunopeptidome, such as polymorphic, cryptic and mutant peptides, can now be explored using novel proteogenomic approaches that combine mass spectrometry and next-generation sequencing.

The ability of sea urchins to destroy kelp forests, leaving large areas stripped of vegetation and covered by sparse calcareous algae is well known. The reduction in active predators of sea urchins combined with their broad diet makes them an important factor in the structuring of subtidal benthic marine systems. In central and northern Chile, the sea urchin Tetrapygus niger is known to reduce the spread of the subtidal kelp Lessonia trabeculata. However, its impact on the different development stages of L. trabeculata has never been quantified or compared to other possible causes of the loss of material. The objective of this study was to quantify the grazing impact of T. niger on L. trabeculata at different stages of development (recruits, juveniles and adults). An exclusion experiment was conducted to evaluate the grazing effect of T. niger on kelp recruitment within a kelp bed, and kelp transplant experiments were conducted to quantify T. niger's impact on the stipes and fronds of juvenile and adult L. trabeculata. Our results showed that under natural sea urchin densities (10 ind. m− 2), T. niger prevented the recruitment of L. trabeculata. Tetrapygus niger completely consumed juvenile plants but only attacked the stipes of adult plants. Tetrapygus niger seems to use different feeding strategies depending on the ontogeny of the plant. Lessonia trabeculata seems unable to defend itself against the impact of intensive grazing by sea urchins, which may be the primary source of mortality of recruits and juveniles of L. trabeculata. However, T. niger's impact on adult plants is limited and shared with other herbivores that graze the fronds, such as fishes and spider crabs.

In a context where injection of antigen (Ag)-specific T cells probably represents the future of leukemia immunotherapy, identification of optimal target Ags is crucial. We therefore sought to discover a reliable marker for selection of the most potent Ags. To this end, (1) we immunized mice against 8 individual Ags: 4 minor histocompatibility Ags (miHAs) and 4 leukemia-associated Ags (LAAs) that were overexpressed on leukemic relative to normal thymocytes; (2) we assessed their ability to reject EL4 leukemic cells; and (3) we correlated the properties of our Ags (and their cognate T cells) with their ability to induce protective antileukemic responses. Overall, individual miHAs instigated more potent antileukemic responses than LAAs. Three features had no influence on the ability of primed T cells to reject leukemic cells: (1) MHC-peptide affinity; (2) the stability of MHC-peptide complexes; and (3) epitope density at the surface of leukemic cells, as assessed using mass spectrometry. The cardinal feature of successful Ags is that they were recognized by high-avidity CD8 T cells that proliferated extensively in vivo. Our work suggests that in vitro evaluation of functional avidity represents the best criterion for selection of Ags, which should be prioritized in clinical trials of leukemia immunotherapy.

Descending inputs to spinal cord neurons in mammals have previously been characterized functionally using microelectrode recording of single neurons, a technique with high spatial and temporal resolution but low yield. Consequently our knowledge about the functional connections between the brain and the spinal cord has been accumulating at a very low pace. Here we describe a high throughput optical recording approach in an ex vivo brainstem-spinal cord preparation of the neonatal mouse that permits screening many spinal neurons simultaneously for synaptic inputs from descending axons. The fluorescent calcium indicator calcium green dextran amine was loaded retrogradely into specific spinal neuron populations, including motoneurons (MNs) of the medial and lateral motor columns and two populations of interneurons with descending axons (dINs) in the ventral funiculus. Focal electrical stimulation of brainstem neuron populations with descending axons generated synaptic responses revealed by transient increases in intracellular calcium concentration in all four populations of spinal neurons. The resultant fluorescence signals could be readily visualized in individual MNs directly through the ventral white matter. In the more deeply located dINs, responses could be readily visualized in individual neurons from the surface of an oblique cut through the spinal cord. The rapid optical investigation of functional connections between brainstem descending neurons and various populations of spinal neurons in the living mammalian preparation should help uncover some of the key features of supraspinal sensory and motor control and provide a valuable tool for examining the re-innervation of spinal neurons by descending axons after spinal cord regeneration.

Summary Despite studies based on deletion or activation of intracellular components of the canonical Wingless related (Wnt) pathway, the role of Wnts in hematolymphopoiesis remains controversial. Using gain-of-function and loss-of-function models, we found that Wnt4 differentially affected diverse subsets of hematopoietic stem and progenitor cells. Bone-marrow and thymic Lin – Sca1 + Kit hi cells (LSKs) were the key targets of Wnt4. In adult mice, Wnt4-induced expansion of Flt3 + bone-marrow LSKs (lymphoid-primed multipotent progenitors) led to a sizeable accumulation of the most immature thymocyte subsets (upstream of β-selection) and a major increase in thymopoiesis. Conversely, Wnt4 –/– neonates showed low frequencies of bone-marrow LSKs and thymic hypocellularity. We provide compelling evidence that Wnt4 activates noncanonical (β-catenin-independent) signaling and that its effects on hematopoietic cells are mainly non-cell-autonomous. Our work shows that Wnt4 overexpression has a unique ability to expand Flt3 + LSKs in adults and demonstrates that noncanonical Wnt signaling regulates thymopoiesis.

Stem cells and immortalized cells have considerable therapeutic potential but present risks of malignant transformation. Cell microencapsulation allows transplantation without immunosuppression. We have developed a method for microencapsulating living cells within covalently cross-linked membranes that are chemically and mechanically extremely resistant. We provide herein direct evidence that these microcapsules can prevent malignant cell dissemination. When 20,000 or more nonencapsulated EL-4 thymoma cells were implanted intraperitoneally in mice, all recipients died with widespread metastasis within 26.3+/-1.0 days. All recipients of 250,000 EL-4 cells microencapsulated in covalently cross-linked membranes were living and disease-free, 150 days post-implantation. Encapsulation in standard microcapsules only slightly delayed the recipient death. Pancreatic islets transplanted using either type of microcapsule presented similar survival. We conclude that microencapsulation in covalently cross-linked membranes prevents malignant cell dissemination.

The STT3 subunit of the oligosaccharyltransferase complex plays a critical role in the N-glycosylation process. From Arabidopsis thaliana to Homo sapiens, two functional STT3 isoforms have been identified, STT3-A and STT3-B. We report that the last transmembrane (TM) segment of STT3-B corresponds to a topogenic determinant that is sufficient for proper integration and orientation of STT3-B C-terminal domain. Notably, the last TM segment of STT3-A and -B isoforms present major differences in amino acid sequence and predicted 3D structure. We also identified a bipartite nuclear targeting sequence in the C-terminal tail of STT3-B that is absent in STT3-A. The latter sequence is sufficient to induce nucleolar localization of a reporter protein. Our results show that STT3-A and -B display two structural differences that may have a drastic influence on their function and might account for the remarkable evolutionary conservation of the two STT3 paralogs.

When presented alone, H7a and HY antigens elicit CD8 T-cell responses of similar amplitude, but H7a totally abrogates the response to HY when both antigens are presented on the same antigen-presenting cell. We found that H7a- and HY-specific T-cell precursors had similar frequencies in nonimmune mice and expressed similar levels of CD5. The H7a-specific CD8 T-cell repertoire harvested at the time of primary response showed highly restricted T-cell receptor (TCR) diversity. Furthermore, T cells specific for H7a and HY expressed equivalent levels of CD8 and TCR and displayed similar tetramer decay rates. The key difference was that anti-H7a T cells exhibited a much more rapid TCR:epitope on-rate than anti-HY T cells. Coupled with evidence that primed CD8 T cells limit the duration of antigen presentation by killing or inactivating antigen-presenting cells, our data support a novel and simple model for immunodomination: the main feature of T cells that exert immunodomination is that, compared with other T cells, they are functionally primed after a shorter duration of antigen presentation.

Chronic exposure to oncostatin M (OM) induces massive thymus-independent extrathymic T-cell development, which takes place solely in the lymph nodes (LN), probably because of the high density of OM receptor on LN postcapillary venules. In contrast to what is observed in other models of extrathymic T-cell development, the proportions of doublenegative (DN), double-positive (DP), and single-positive (SP) T cells in the OM LN reproduce those found in a thymus, and the T-cell receptor (TCR) repertoire of the SP cells is diversified. Analyses of T-cell development in hematopoietic chimeras expressing MHC class I only on hematopoietic or non-hematopoietic cells showed that MHC class I on hematopoietic cells was sufficient for positive selection of a diversified repertoire of CD8 T cells. However, studies of TCR-transgenic mice revealed that the hierarchy of TCR clonotype selection is not the same as in the thymus. Even though there is no cortico-medullary segregation in the OM LN, negative selection of T cells bearing the 2C and the anti-H-Y TCR was very effective. The progeny of the thymic-independent differentiation pathway regulated by OM is polyclonal in terms of V usage, exhibits an activated-memory phenotype, and displays a rapid turnover rate. Following TCR ligation, extrathymic T cells begin to proliferate earlier than thymus-derived T cells, but their expansion is limited by a high apoptosis rate. This model provides a unique opportunity to elucidate critical interactions between T cell progenitors and stromal cells, that is, the essence of a primary T lymphoid organ.

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins.

The antigen-binding site of most major histocompatibility complex (MHC) class I and II antigens is occupied by self-peptides that are derived from the proteolysis of endogenous proteins following instructions provided by the molecules of the MHC themselves. Together with MHC proteins, self-peptides define our immunological self and shape the repertoire of both T cells that recognize "nonself," and NK cells that may recognize "no self." Endogenous proteins of all cell compartments (nucleus, cytosol, organelles, surface membrane) can yield self peptides whose expression may be either ubiquitous or lineage-specific. Their expression allows the binary recognition mechanism of T and NK cells to check the integrity of the cell genome. A better understanding of the molecular bases of the distinction between self and nonself permits us to anticipate the possibility of modifying their expression and/or their recognition in order to: (i) make the nonself acceptable as self, thereby establishing specific transplantation tolerance, (ii) reestablish tolerance of the self lost in autoimmune diseases, and (iii) induce the rejection as nonself of neoplastic cells. These objectives are particularly pertinent to the area of bone marrow transplantation, where the ultimate goal is aimed at modulating host cell allorecognition in such a way as to both potentiate the graft-versus-leukemia reaction and prevent GVHD.

This short review summarizes a number of findings related to the coordination of activity in muscles of the same limb or of different limbs during real locomotion on a treadmill of intact and spinal cats and during ‘fictive’ locomotion in paralyzed cats. Firstly, it is shown that a number of discharge characteristics can be observed in the three conditions. Secondly, it is shown that tonic proprioceptive inputs from the hip and shoulder have powerful effects on the overall locomotor structure. Phasic proprioceptive and cutaneous inputs also exert phase-dependent effects on the different muscle group during locomotion. Thirdly, it is shown that the activity of the medullary reticular formation is compatible with its potential role in interlimb coordination.

Allogeneic stem cell transplantation (allo-SCT) can induce remission in patients with hematological malignancies due to graft-versus-tumor (GVT) responses. This immune-mediated anti-tumor effect, however, is often accompanied by detrimental graft-versus-host disease (GVHD). Both GVT and GVHD are mediated by minor histocompatibility antigen (MiHA)-specific T cells recognizing peptide products from polymorphic genes that differ between recipient and donor. In this study, we evaluated whether mismatches in a panel of seventeen MiHA are associated with clinical outcome after partial T cell-depleted allo-SCT. Comprehensive statistical analysis revealed that DNA mismatches for one or more autosomal-encoded MiHA was associated with increased relapse-free survival in sibling transplants, (P =0.04), particularly in patients suffering from multiple myeloma (P =0.02). Moreover, mismatches for the ubiquitous Y chromosome-derived MiHA resulted in a higher incidence of acute GVHD (grade 3–4; P =0.004), while autosomal MiHA mismatches, ubiquitous or restricted to hematopoietic cells, were not associated with severe GVHD. Finally, we demonstrated considerable differences between MiHA in their capability to induce in vivo T cell responses using dual-color tetramer analysis of peripheral blood samples collected post-SCT. Importantly, detection of MiHA-specific T cell responses was associated with improved relapse-free survival in sibling transplants (P =0.01). Our findings provide a rationale to further boost GVT immunity towards autosomal MiHA with a hematopoietic restriction to improve outcome after HLA-matched allo-SCT.

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule GranzymeB, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Cytogenetic analyses were carried out in a young Haitian immigrant with acquired immunodeficiency syndrome and concomitant Burkitt's-like lymphoma associated with massive bone marrow infiltration. A characteristic 14q+ abnormality was found in all bone marrow cells examined. Although chromosome abnormality involving band 8q24 was not evident in all the cells examined, some karyotypes show that the typical t(8;14)(q24;q32) is most probably present. No other complex rearrangements could be identified. This is the first report of concomitant acquired immunodeficiency syndrome and Burkitt's-like lymphoma in the Haitian community. Our cytogenetic findings provide further evidence for the role of specific chromosomal rearrangements in Burkitt's-like lymphoma oncogenesis in the setting of acquired immunodeficiency syndrome.