Inflammatory CD4/CD8 double-positive human T cells arise from reactive CD8 T cells and are sufficient to mediate GVHD pathology.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4+/CD8+ double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.

Characterization of the HHV-6B U20 Immunoevasin.

Roseoloviruses (human herpesvirus 6A [HHV-6A], -6B, and -7) infect >90% of the human population during early childhood and are thought to remain latent or persistent throughout the life of the host. As such, these viruses are among the most pervasive and stealthy of all viruses; they must necessarily excel at escaping immune detection throughout the life of the host, and yet, very little is known about how these viruses so successfully escape host defenses. Here, we characterize the expression, trafficking, and posttranslational modifications of the HHV6B U20 gene product, which is encoded within a block of genes unique to the roseoloviruses. HHV-6B U20 trafficked slowly through the secretory system, receiving several posttranslational modifications to its N-linked glycans, indicative of surface-expressed glycoproteins, and eventually reaching the cell surface before being internalized. Interestingly, U20 is also phosphorylated on at least one Ser, Thr, or Tyr residue. These results provide a framework to understand the role(s) of U20 in evading host defenses. IMPORTANCE The roseolovirus U20 proteins are virus-encoded integral membrane glycoproteins possessing class I major histocompatibility complex (MHC)-like folds. Surprisingly, although U20 proteins from HHV-6A and -6B share 92% identity, recent studies ascribe different functions to HHV6A U20 and HHV6B U20. HHV6A U20 was shown to downregulate NKG2D ligands, while HHV6B U20 was shown to inhibit tumor necrosis factor alpha (TNF-α)-induced apoptosis during nonproductive infection with HHV6B (E. Kofod-Olsen, K. Ross-Hansen, M. H. Schleimann, D. K. Jensen, et al., J Virol 86:11483-11492, 2012, https://doi.org/10.1128/jvi.00847-12; A. E. Chaouat, B. Seliger, O. Mandelboim, D. Schmiedel, Front Immunol 12:714799, 2021, https://doi.org/10.3389/fimmu.2021.714799). Here, we have performed cell biological and biochemical characterization of the trafficking, glycosylation, and posttranslational modifications occurring on HHV6B U20.

iNKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that interact with key antigen-presenting cells to modulate adaptive T-cell responses in ways that can either promote protective immunity, or limit pathological immune activation. Understanding the immunological networks engaged by iNKT cells to mediate these opposing functions is a key pre-requisite to effectively using iNKT cells for therapeutic applications. Using a human umbilical cord blood xenotransplantation model, we show here that co-transplanted allogeneic CD4+ iNKT cells interact with monocytes and T cells in the graft to coordinate pro-hematopoietic and immunoregulatory pathways. The nexus of iNKT cells, monocytes, and cord blood T cells led to the release of cytokines (IL-3, GM-CSF) that enhance hematopoietic stem and progenitor cell activity, and concurrently induced PGE2-mediated suppression of T-cell inflammatory responses that limit hematopoietic stem and progenitor cell engraftment. This resulted in successful long-term hematopoietic engraftment without pretransplant conditioning, including multi-lineage human chimerism and colonization of the spleen by antibody-producing human B cells. These results highlight the potential for using iNKT cellular immunotherapy to improve rates of hematopoietic engraftment independently of pretransplant conditioning.

Different Human Immune Lineage Compositions Are Generated in Non-Conditioned NBSGW Mice Depending on HSPC Source.

NBSGW mice are highly immunodeficient and carry a hypomorphic mutation in the c-kit gene, providing a host environment that supports robust human hematopoietic expansion without pre-conditioning. These mice thus provide a model to investigate human hematopoietic engraftment in the absence of conditioning-associated damage. We compared transplantation of human CD34+ HSPCs purified from three different sources: umbilical cord blood, adult bone marrow, and adult G-CSF mobilized peripheral blood. HSPCs from mobilized peripheral blood were significantly more efficient (as a function of starting HSPC dose) than either cord blood or bone marrow HSPCs at generating high levels of human chimerism in the murine blood and bone marrow by 12 weeks post-transplantation. While T cells do not develop in this model due to thymic atrophy, all three HSPC sources generated a human compartment that included B lymphocytic, myeloid, and granulocytic lineages. However, the proportions of these lineages varied significantly according to HSPC source. Mobilized blood HSPCs produced a strikingly higher proportion of granulocyte lineage cells (~35% as compared to ~5%), whereas bone marrow HSPC output was dominated by B lymphocytic cells, and cord blood HSPC output was enriched for myeloid lineages. Following transplantation, all three HSPC sources showed a shift in the CD34+ subset towards CD45RA+ progenitors along with a complete loss of the CD45RA-CD49f+ long-term HSC subpopulation, suggesting this model promotes mainly short-term HSC activity. Mice transplanted with cord blood HSPCs maintained a diversified human immune compartment for at least 36 weeks after the primary transplant, although mice given adult bone marrow HSPCs had lost diversity and contained only myeloid cells by this time point. Finally, to assess the impact of non-HSPCs on transplantation outcome, we also tested mice transplanted with total or T cell-depleted adult bone marrow mononuclear cells. Total bone marrow mononuclear cell transplants produced significantly lower human chimerism compared to purified HSPCs, and T-depletion rescued B cell levels but not other lineages. Together these results reveal marked differences in engraftment efficiency and lineage commitment according to HSPC source and suggest that T cells and other non-HSPC populations affect lineage output even in the absence of conditioning-associated inflammation.

Early T Cell Activation Metrics Predict Graft-versus-Host Disease in a Humanized Mouse Model of Hematopoietic Stem Cell Transplantation.

Acute graft-versus-host disease (GVHD) is a frequent complication of hematopoietic transplantation, yet patient risk stratification remains difficult, and prognostic biomarkers to guide early clinical interventions are lacking. We developed an approach to evaluate the potential of human T cells from hematopoietic grafts to produce GVHD. Nonconditioned NBSGW mice transplanted with titrated doses of human bone marrow developed GVHD that was characterized by widespread lymphocyte infiltration and organ pathology. Interestingly, GVHD was not an inevitable outcome in our system and was influenced by transplant dose, inflammatory status of the host, and type of graft. Mice that went on to develop GVHD showed signs of rapid proliferation in the human T cell population during the first 1-3 wk posttransplant and had elevated human IFN-γ in plasma that correlated negatively with the expansion of the human hematopoietic compartment. Furthermore, these early T cell activation metrics were predictive of GVHD onset 3-6 wk before phenotypic pathology. These results reveal an early window of susceptibility for pathological T cell activation following hematopoietic transplantation that is not simply determined by transient inflammation resulting from conditioning-associated damage and show that T cell parameters during this window can serve as prognostic biomarkers for risk of later GVHD development.

Adoptively transferred Vγ9Vδ2 T cells show potent antitumor effects in a preclinical B cell lymphomagenesis model.

A central issue for adoptive cellular immunotherapy is overcoming immunosuppressive signals to achieve tumor clearance. While γδ T cells are known to be potent cytolytic effectors that can kill a variety of cancers, it is not clear whether they are inhibited by suppressive ligands expressed in tumor microenvironments. Here, we have used a powerful preclinical model where EBV infection drives the de novo generation of human B cell lymphomas in vivo, and autologous T lymphocytes are held in check by PD-1/CTLA-4-mediated inhibition. We show that a single dose of adoptively transferred Vδ2+ T cells has potent antitumor effects, even in the absence of checkpoint blockade or activating compounds. Vδ2+ T cell immunotherapy given within the first 5 days of EBV infection almost completely prevented the outgrowth of tumors. Vδ2+ T cell immunotherapy given more than 3 weeks after infection (after neoplastic transformation is evident) resulted in a dramatic reduction in tumor burden. The immunotherapeutic Vδ2+ T cells maintained low cell surface expression of PD-1 in vivo, and their recruitment to tumors was followed by a decrease in B cells expressing PD-L1 and PD-L2 inhibitory ligands. These results suggest that adoptively transferred PD-1lo Vδ2+ T cells circumvent the tumor checkpoint environment in vivo.

The effect of obesity on acute kidney injury after cardiac surgery.

OBJECTIVE: Postoperative acute kidney injury is a frequent and serious consequence of cardiac surgery. We undertook to investigate the association of obesity and the risk of acute kidney injury development after cardiac surgery. METHODS: A total of 432 patients who underwent cardiac surgery with cardiopulmonary bypass between October 2009 and August 2010 were included in the final retrospective analysis. Obesity was defined as body mass index 30 kg/m(2) or greater. Acute kidney injury was defined as a creatinine increase of 25% or more from baseline at 48 hours after surgery. RESULTS: The overall incidence of acute kidney injury was 29.9% (n = 129). There was an increased incidence of postoperative renal impairment in the obese versus nonobese cohort; however, this was not statistically significant (39% vs 25.9%, P = .007). Multivariate logistic regression revealed that body mass index 30 kg/m(2) or greater was independently associated with the development of postoperative acute kidney injury (odds ratio [OR], 2.12; 95% confidence interval [CI], 1.27-3.54; P = .004), as were age (OR, 0.98; 95% CI, 0.96-1.0; P = .04) and cardiopulmonary bypass time (OR, 0.99; 95% CI, 0.98-1.0; P = .048). CONCLUSIONS: Obesity with body mass index 30 kg/m(2) or greater is independently associated with an increased risk of acute kidney injury after cardiac surgery. Further understanding of the molecular basis of this association is critical to the design of preventative strategies.

Roseoloviruses and their modulation of host defenses.

Human cytomegalovirus (HCMV), the prototypical human ß-herpesvirus, encodes approximately 40 known gene products that function to subvert our host defense mechanisms. From HCMV, we have learned about interferon signaling, cytokine function, chemokine signaling, natural killer (NK) cells' cytotoxicity toward tumors and virus-infected cells, antigen processing and presentation, and protective initiation of the apoptotic signaling cascade. With each successive discovery of novel host evasion mechanism encoded by the cytomegaloviruses, we illuminate what these herpesviruses have learned over the course of their 100 MYr-long evolution with their hosts. As much as we have learned from HCMV, the other members of the human ß-herpesvirus family, HHV-6 and HHV-7, are closely-related and yet largely unexplored. These viruses likely have much yet to teach us.

Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the µ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining µ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

Expression of CD1c enhances human invariant NKT cell activation by α-GalCer.

Invariant natural killer T (iNKT) cells are innate T lymphocytes that specifically recognize α-linked glycosphingolipids (α-GSLs) as antigens presented by CD1d molecules. Activating iNKT cells by administering α-GSLs improves disease outcomes in murine cancer models and, thus, there is great interest in the clinical potential of these lipids for treating human cancers. However, humans possess several other CD1 isoforms that are not present in mice and it is not clear whether these CD1 molecules, which also bind lipids, affect human iNKT cell responses. We demonstrate here that CD1c, which is co-expressed with CD1d on blood dendritic cells and on a fraction of B cells, is able to present α-galactosylceramide (α-GalCer) as a weak agonist to human iNKT cells, and that the presence of CD1c synergistically enhances α-GalCerdependent activation of iNKT cells by CD1d. Primary human B cells expressing CD1c induced stronger iNKT cell responses to α-GalCer than the CD1c- subset, and an antibody against CD1c inhibited iNKT cell cytokine secretion. These results suggest that therapeutic activation of human iNKT cells by α-GSLs will be driven preferentially by CD1c+ cell types. Thus, B cell neoplasias that co-express CD1c and CD1d may be particularly susceptible to α-GSL therapy, and cancer vaccines using α-GSLs as adjuvants may be most effective when presented by CD1c+ antigen-presenting cells.

The human herpesvirus-7 (HHV-7) U21 immunoevasin subverts NK-mediated cytoxicity through modulation of MICA and MICB.

Herpesviruses have evolved numerous immune evasion strategies to facilitate establishment of lifelong persistent infections. Many herpesviruses encode gene products devoted to preventing viral antigen presentation as a means of escaping detection by cytotoxic T lymphocytes. The human herpesvirus-7 (HHV-7) U21 gene product, for example, is an immunoevasin that binds to class I major histocompatibility complex molecules and redirects them to the lysosomal compartment. Virus infection can also induce the upregulation of surface ligands that activate NK cells. Accordingly, the herpesviruses have evolved a diverse array of mechanisms to prevent NK cell engagement of NK-activating ligands on virus-infected cells. Here we demonstrate that the HHV-7 U21 gene product interferes with NK recognition. U21 can bind to the NK activating ligand ULBP1 and reroute it to the lysosomal compartment. In addition, U21 downregulates the surface expression of the NK activating ligands MICA and MICB, resulting in a reduction in NK-mediated cytotoxicity. These results suggest that this single viral protein may interfere both with CTL-mediated recognition through the downregulation of class I MHC molecules as well as NK-mediated recognition through downregulation of NK activating ligands.

Insight into the mechanism of human herpesvirus 7 U21-mediated diversion of class I MHC molecules to lysosomes.

The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules.

The ER-lumenal domain of the HHV-7 immunoevasin U21 directs class I MHC molecules to lysosomes.

Like all members of the herpesvirus family, human herpesvirus-7 has evolved mechanisms to evade immune detection. The human herpesvirus-7 gene product U21 encodes an immunoevasin that binds to class I major histocompatibility complex molecules and diverts them to a lysosomal compartment. Here we show that the cytoplasmic tail of U21, although sufficient to sequester a heterologous membrane protein (CD4 chimera), has no effect on U21's ability to redirect class I major histocompatibility complex molecules to lysosomes. Instead, the ER-lumenal domain of U21 is sufficient to redirect class I major histocompatibility complex molecules to the lysosomal compartment. These observations demonstrate a novel viral immunoevasive mechanism for U21, and implicate the ER-lumenal domain of a type I transmembrane protein in lysosomal sorting.