Functional consequences of genetic polymorphisms in the NKG2D receptor signaling pathway and putative gene interactions

NKG2D (NK group 2, member D) is an activating natural killer (NK) receptor, which is expressed on NK and CD8+ T cells. On NK cells, NKG2D elicits cytotoxicity and release of cytokines. On CD8+ T cells, it functions as a co-stimulatory molecule. The receptor recognizes several ligands including the major histocompatibility complex (MHC) class I chain-related molecules A (MICA) and B (MICB) as well as the UL16-binding proteins (ULBP). The diversity of NKG2D ligands is further increased by a high degree of genetic variability of the ligands. Recently, an amino acid exchange from valine to methionine at position 129 in MICA has been found to be associated with the outcome of allogeneic hematopoietic stem cell transplantation (HSCT), and the functional consequences of this specific genetic variation have been elucidated. The clinical associations found after HSCT were explainable by the functional differences of the MICA-129 variants. Herein, we discuss how the genetic polymorphisms of NKG2D ligands and NKG2D itself interact and may affect the outcome of HSCT and the susceptibility to other diseases, which have been associated with polymorphisms in the NKG2D signaling pathway

Case Report: Graft Versus Tumor Effect After Non-Myeloablative Allogeneic Stem-Cell Transplantation in a Patient With Brentuximab-Vedotin Refractory Sezary Syndrome

Sezary Syndrome (SS) is a rare leukemic variant of primary cutaneous T-cell lymphoma. Relapsed or refractory disease is generally considered incurable by conventional therapeutic approaches, although durable responses can be achieved with novel monoclonal antibodies. Allogeneic hematopoietic stem cell transplantation (alloHSCT) may have potential value by inducing graft vs-lymphoma (GvL) effects, but there is currently no consensus regarding the timing of alloHSCT or type of conditioning regimen. Here we present the case of a male patient who achieved a complete remission (CR) of primary refractory SS after non-myeloablative alloHSCT. Patient: Two years prior to HSCT, the patient had been refractory to CHOEP-based chemotherapy, interferon, extracorporeal photopheresis (ECP), and bexarotene. Directly prior to alloHSCT brentuximab-vedotin (BV) was applied resulting in a partial remission of the skin compartment and overall in a stable disease. Prior to HSCT, flow cytometry of the bone marrow and peripheral blood showed an infiltration with T-cells positive for CD5, CD4, low CD3, low CD2 and negative for CD7, CD38, HLA-DR and CD8. The trephine biopsy showed a 7% infiltration of SS cells. The CD4:CD8 ratio in peripheral blood (pb) was massively increased at 76.67, with 63.5% of white blood cells expressing a SS immune phenotype. The conditioning regimen included 30 mg/m2 fludarabine on days -5, -4 and -3 and total body irradiation with 2 Gy on day -1. Immunosuppression consisted of cyclosporine A from day-1 and mycophenolate mofetil from day 0. The patient received 6.55x106 CD34+ cells and 1.11x108 CD3+ cells/kg body weight. Bone marrow evaluation on day 28 still showed persistent SS cells by flow cytometry. After tapering immunosuppression until day 169, the CD4:CD8 ratio in pb normalized. CR was documented on day 169 after alloHSCT and is now ongoing for almost 3 years after alloHSCT. Conclusions: We confirm that an alloHSCT can be a curative option for refractory patients with SS. The achievement of a CR after tapering the immunosuppressive therapy indicates a significant role of the GvL effect. In present treatment algorithms for patients with SS, the timing of an alloHSCT and the intensity of conditioning should be further explored

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

BACKGROUND: Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). RESULTS: We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. CONCLUSION: Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells. REVIEWERS: This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter

CD8+-T Cells With Specificity for a Model Antigen in Cardiomyocytes Can Become Activated After Transverse Aortic Constriction but Do Not Accelerate Progression to Heart Failure

Heart failure due to pressure overload is frequently associated with inflammation. In addition to inflammatory responses of the innate immune system, autoimmune reactions of the adaptive immune system appear to be triggered in subgroups of patients with heart failure as demonstrated by the presence of autoantibodies against myocardial antigens. Moreover, T cell-deficient and T cell-depleted mice have been reported to be protected from heart failure induced by transverse aortic constriction (TAC) and we have shown recently that CD4+-helper T cells with specificity for an antigen in cardiomyocytes accelerate TAC-induced heart failure. In this study, we set out to investigate the potential contribution of CD8+-cytotoxic T cells with specificity to a model antigen (ovalbumin, OVA) in cardiomyocytes to pressure overload-induced heart failure. In 78% of cMy-mOVA mice with cardiomyocyte-specific OVA expression, a low-grade OVA-specific cellular cytotoxicity was detected after TAC. Adoptive transfer of OVA-specific CD8+-T cells from T cell receptor transgenic OT-I mice before TAC did not increase the risk of OVA-specific autoimmunity in cMy-mOVA mice. After TAC, again 78% of the mice displayed an OVA-specific cytotoxicity with on average only a three-fold higher killing of OVA-expressing target cells. More CD8+ cells were present after TAC in the myocardium of cMy-mOVA mice with OT-I T cells (on average 17.5/mm2) than in mice that did not receive OVA-specific CD8+-T cells (3.6/mm2). However, the extent of fibrosis was similar in both groups. Functionally, as determined by echocardiography, the adoptive transfer of OVA-specific CD8+-T cells did not significantly accelerate the progression from hypertrophy to heart failure in cMy-mOVA mice. These findings argue therefore against a major impact of cytotoxic T cells with specificity for autoantigens of cardiomyocytes in pressure overload-induced heart failure

Salvage Therapy With Polatuzumab Vedotin, Bendamustine, and Rituximab Prior to Allogeneic Hematopoietic Transplantation in Patients With Aggressive Lymphomas Relapsing After Therapy With Chimeric Antigen Receptor T-Cells—Report on Two Cases

Up to 60% of patients with aggressive B-cell lymphoma who receive chimeric antigen receptor (CAR) T-cell therapy experience treatment failure and subsequently have a poor prognosis. Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains a potentially curative approach for patients in this situation. Induction of a deep response prior to alloHSCT is crucial for long-term outcomes, but the optimal bridging strategy following relapse after CAR T-cell therapy has not yet been established. Polatuzumab vedotin, an antibody drug conjugate targeting CD79b, is a novel treatment option for use in combination with rituximab and bendamustine (Pola-BR) in relapsed or refractory disease. Patients: We report two heavily pretreated patients with primary refractory diffuse large Bcell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) respectively who relapsed after therapy with CAR T-cells with both nodal and extranodal manifestations of the disease. After application of three courses of Pola-BR both patients achieved a complete metabolic remission. Both patients underwent alloHSCT from a human leukocyte antigen (HLA)-mismatched donor following conditioning with busulfan and fludarabine and are disease free 362 days and 195 days after alloHSCT respectively. We conclude that Pola-BR can be an effective bridging therapy before alloHSCT of patients relapsing after CAR T-cell therapy. Further studies will be necessary to define the depth and durability of remission of this salvage regimen before alloHSCT

Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D

Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1−/− mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amount

Diversity of Staphylococcus aureus Isolates in European Wildlife

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle- associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock- associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation

Exploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone

This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe

Phylodynamic signatures in the emergence of community-associated MRSA

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers