Risk Factors for Severe Acute Graft-versus-Host Disease in Donor Graft Composition.

Acute graft-versus-host disease (aGVHD) is 1 of the main major complications of post-hematopoietic stem cell transplantation (HSCT). Identifying patients at risk of severe aGVHD may lead to earlier intervention and treatment, resulting in increased survival and a better quality of life. We aimed to identify biomarkers in donor grafts and patient plasma around the time of transplantation that might be predictive of aGVHD development. We build on our previously published methods by using multiplex assays and multicolor flow cytometry. We identified 5 easily assessable cellular markers in donor grafts that combined could potentially be used to calculate risk for severe aGVHD development. Most noteworthy are the T cell subsets expressing IL-7 receptor-α (CD127) and PD-1. Additionally, we identified a potential role for elevated tumor necrosis factor-α levels in both graft and patient before HSCT in development of aGVHD.

Exhausted Tumor-infiltrating CD39+CD103+ CD8+ T Cells Unveil Potential for Increased Survival in Human Pancreatic Cancer.

In pancreatic ductal adenocarcinoma, the infiltration of CD8+ T cells within the tumor microenvironment correlates with a favorable prognosis. However, a significant proportion of tumor-infiltrating T cells become trapped within the desmoplastic stroma and lack tumor reactivity. Here, we explored different T-cell subsets in pancreatic tumors and adjacent tissues. We identified a subset of CD8+ T cells, double positive (DP) for CD39 and CD103 in pancreatic tumors, which has recently been described to display tumor reactivity in other types of solid tumors. Interestingly, DP CD8+ T cells preferentially accumulated in central tumor tissues compared with paired peripheral tumor and adjacent non-tumor tissues. Consistent with an antigen encounter, DP CD8+ T cells demonstrated higher proliferative rates and displayed an exhausted phenotype, characterized by elevated expression of PD-1 and TIM-3, compared with CD39-CD103- CD8+ T cells. In addition, DP CD8+ T cells exhibited higher expression levels of the tissue trafficking receptors CCR5 and CXCR6, while displaying lower levels of CXCR3 and CXCR4. Importantly, a high proportion of DP CD8+ T cells is associated with increased patient survival. These findings suggest that DP CD8+ T cells with a phenotype reminiscent of that of tumor-reactive T cells are present in pancreatic tumors. The abundance of DP CD8+ T cells could potentially aid in selecting patients for pancreatic cancer immunotherapy trials. SIGNIFICANCE: Patients with pancreatic cancer with a high proportion of CD39+CD103+ CD8+ T cells exhibiting a tumor-reactive phenotype have improved survival rates, suggesting their potential utility in selecting candidates for immunotherapy trials.

Labour promotes systemic mobilisation of monocytes, T cell activation and local secretion of chemotactic factors in the intervillous space of the placenta.

During pregnancy, maternal blood circulates through the intervillous space of the placenta and the reciprocal interactions between foetal tissues and maternal immune cells makes the intervillous space a unique immunological niche. Labour is characterised by a proinflammatory response in the myometrium, but the relationship between local and systemic changes during the onset of labour remains elusive. We here aimed to investigate how the systemic and intervillous circulatory systems are affected during labour from an immunological point of view. We report that the proportion of monocytes is dramatically higher in peripheral (PB), intervillous blood (IVB) and decidua in labouring (n = 14) compared to non-labouring women (n = 15), suggesting that labour leads to both a systemic and local mobilisation of monocytes. Labour was associated with a relative increase of effector memory T cells in the intervillous space compared to the periphery, and MAIT cells and T cells showed an elevated expression of activation markers both in PB and IVB. Intervillous monocytes consisted to a higher degree of CD14+CD16+ intermediate monocytes compared to peripheral monocytes, independently of mode of delivery, and displayed an altered phenotypic expression pattern. A proximity extension assay analysis of 168 proteins revealed that several proteins associated to myeloid cell migration and function, including CCL2 and M-CSF, were upregulated in IVB plasma in labouring women. Thus, the intervillous space could be a bridging site for the communication between the placenta and the periphery, which contribute to monocyte mobilisation and generation of inflammatory reactions during spontaneous labour.

MAIT Cells in Health and Disease.

Mucosal-associated invariant T (MAIT) cells are a newly described subset of T cells that are found in the blood and are enriched in many tissues, particularly in the liver. MAIT cells express a semi-invariant T cell receptor restricted by the MHC class I-related (MR1) molecule. MAIT cells are activated in a MR1-dependent manner in response to microbial-derived riboflavin metabolites which leads to rapid effector functions, but they can also be activated in a MR1-independent manner by cytokines and viruses. The use of mice models and MR1 tetramers, among other recent methodological advances, have provided more insight into the development, mode of activation, characterization in different diseases and tissues of MAIT cells. In this chapter, we provide an overview of MAIT cells and yet remaining questions about their potential therapeutic role.

Isolation of Immune Cells from Placental Tissues and Phenotypic and Functional Analysis of MAIT Cells.

The placenta is an immunological paradox since maternal immune cells infiltrating placental tissues need to be tolerant toward the fetus but still retain immunity against potential infections. This makes the placenta an interesting tissue for studying immunological processes. Mucosal-associated invariant T (MAIT) cells are a subset of T cells that respond to bacterially derived metabolites of riboflavin synthesis. Upon activation, MAIT cells respond by secretion of inflammatory cytokines and by directed killing of infected cells by the use of granzymes and perforin. In this protocol, we describe methods for the isolation of immune cells from the placental intervillous space and adjacent tissues such as the umbilical cord, decidua parietalis, and decidua basalis. We further describe how to stimulate MAIT cells in mixed cell suspensions of mononuclear cells with bacteria, and how to analyze the phenotypic and functional responses with flow cytometry.

MAIT Cells at the Fetal-Maternal Interface During Pregnancy.

One of the main functions of the human placenta is to provide a barrier between the fetal and maternal blood circulations, where gas exchange and transfer of nutrients to the developing fetus take place. Despite being a barrier, there is a multitude of crosstalk between maternal immune cells and fetally derived semi-allogeneic trophoblast cells. Therefore, the maternal immune system has a difficult task to both tolerate the fetus but at the same time also defend the mother and the fetus from infections. Mucosal-associated invariant T (MAIT) cells are an increasingly recognized subset of T cells with anti-microbial functions that get activated in the context of non-polymorphic MR1 molecules, but also in response to inflammation. MAIT cells accumulate at term pregnancy in the maternal blood that flows into the intervillous space inside the placenta. Chemotactic factors produced by the placenta may be involved in recruiting and retaining particular immune cell subsets, including MAIT cells. In this Mini-Review, we describe what is known about MAIT cells during pregnancy and discuss the potential biological functions of MAIT cells at the fetal-maternal interface. Since MAIT cells have anti-microbial and tissue-repairing functions, but lack alloantigen reactivity, they could play an important role in protecting the fetus from bacterial infections and maintaining tissue homeostasis without risks of mediating harmful responses toward semi-allogenic fetal tissues.

Mature naïve B cells are retained in the placental intervillous blood and positively associate with specific chemokines in full-term healthy pregnancy.

PROBLEM: Circulating B-cell numbers are lower during pregnancy compared with non-pregnant women, but the underlying reasons for this are unknown. Pregnancy-related hormones could influence B-cell lymphopoiesis in the bone marrow, but B cells may also be recruited to the placenta. To investigate the latter, we examined whether the proportions of total B cells and B cells at different maturational stages in placental intervillous blood (IVB) differ compared with peripheral blood (PB). METHOD OF STUDY: From 23 paired samples of PB and IVB following full-term healthy pregnancies, total B cells and immature/transitional, mature/naïve, and memory B cells were identified by flow cytometry. Chemokine levels in blood were analyzed using a Luminex assay. Placental explant-derived supernatant was assayed for B-cell chemotactic activity. RESULTS: The proportions of total B cells and mature/naïve B cells were significantly higher in IVB relative to PB, while the fractions of immature/transitional cells and memory B cells were higher in PB. Multivariate factor analysis demonstrated that a specific chemokine profile in IVB, including CCL20, positively associated with higher proportions of mature/naïve B cells in the intervillous space. All B cells expressed CCR6, the corresponding receptor for CCL20, but the intensity of CCR6 expression was significantly higher in mature/naïve B cells relative to immature/transitional B cells. Migration assays showed that placental explant-derived supernatants attract B cells. CONCLUSION: These results indicate that B cells, and mature/naïve B cells in particular, are retained in the intervillous blood in response to certain chemokines produced by the placenta during late healthy pregnancy.

Recruitment of MAIT Cells to the Intervillous Space of the Placenta by Placenta-Derived Chemokines.

The intervillous space of the placenta is a part of the fetal-maternal interface, where maternal blood enters to provide nutrients and gas exchange. Little is known about the maternal immune cells at this site, which are in direct contact with fetal tissues. We have characterized the T cell composition and chemokine profile in paired intervillous and peripheral blood samples from healthy mothers giving birth following term pregnancies. Mucosal-associated invariant T (MAIT) cells and effector memory (EM) T cells were enriched in the intervillous blood compared to peripheral blood, suggesting that MAIT cells and other EM T cells home to the placenta during pregnancy. Furthermore, pregnant women had lower proportions of peripheral blood MAIT cells compared to non-pregnant women. The levels of several chemokines were significantly higher in intervillous compared to peripheral blood, including macrophage migration inhibitory factor (MIF), CXCL10, and CCL25, whereas CCL21, CCL27 and CXCL12 were lower. Migration assays showed that MAIT cells and EM T cells migrated toward conditioned medium from placental explants. A multivariate factor analysis indicated that high levels of MIF and CCL25 were associated with high proportions of MAIT cells in intervillous blood. Blocking of MIF or a combination of MIF, CCL25, and CCL20 in migration assays inhibited MAIT cell migration toward placenta conditioned medium. Finally, MAIT cells showed migratory capacities toward recombinant MIF. Together, these findings indicate that term placental tissues attract MAIT cells, and that this effect is at least partly mediated by MIF.

Maternal Adaptive Immune Cells in Decidua Parietalis Display a More Activated and Coinhibitory Phenotype Compared to Decidua Basalis.

The maternal part of the placenta, the decidua, consists of maternal immune cells, decidual stromal cells, and extravillous fetal trophoblasts. In a successful pregnancy, these cell compartments interact to provide an intricate balance between fetal tolerance and antimicrobial defense. These processes are still poorly characterized in the two anatomically different decidual tissues, basalis and parietalis. We examined immune cells from decidua basalis and parietalis from term placentas (n = 15) with flow cytometry. By using multivariate discriminant analysis, we found a clear separation between the two decidual compartments based on the 81 investigated parameters. Decidua parietalis lymphocytes displayed a more activated phenotype with a higher expression of coinhibitory markers than those isolated from basalis and contained higher frequencies of T regulatory cells. Decidua basalis contained higher proportions of monocytes, B cells, and mucosal-associated invariant T (MAIT) cells. The basalis B cells were more immature, and parietalis MAIT cells showed a more activated phenotype. Conventional T cells, NK cells, and MAIT cells from both compartments potently responded with the production of interferon-γ and/or cytotoxic molecules in response to stimulation. To conclude, leukocytes in decidua basalis and parietalis displayed remarkable phenotypic disparities, indicating that the corresponding stromal microenvironments provide different immunoregulatory signals.

Mucosal-Associated Invariant T Cells Display a Poor Reconstitution and Altered Phenotype after Allogeneic Hematopoietic Stem Cell Transplantation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells which are important in the defense against certain bacteria and yeast. The reconstitution of MAIT cells after allogeneic hematopoietic stem cell transplantation (HSCT) is not known. We investigated MAIT cell phenotype and function in 17 patients devoid of relapse and severe graft-versus-host disease (GvHD) in paired samples collected 1-2, 3-6, 12, and 24 months after transplantation. Data were compared to 17 healthy controls (HC), as well as 22 patients with acute GvHD grade 2-3. The frequency of MAIT cells within CD3+ cells was approximately 10-fold lower than in HC and did not increase over the 2 years following HSCT. MAIT cells in HSCT patients displayed an elevated expression of CD69 and intracellular granzyme B and were predominantly composed of CD4/CD8 double-negative cells. The expression of PD-1 on MAIT cells was low and did not change during the observational time, whereas the CD3+CD161dim/negTCRVα7.2dim/neg cells (non-MAIT T cells) displayed a high expression early after HSCT that decreased to normal levels at 24 months. MAIT cells collected 2-6 months post-HSCT showed an impaired IFN-γ and perforin response after bacterial stimulation, but the response was restored at 24 months. Patients with acute GvHD had similar proportions of MAIT cells as patients with grade 0-1, but consisted mainly of CD8+ cells. Finally, MAIT cells were more sensitive to cyclosporine A and sirolimus than non-MAIT T cells. To conclude, MAIT cell reconstitution following HSCT is deficient compared to non-MAIT T cells and GvHD grade ≥2 is not correlated with MAIT cell frequency. MAIT cell functionality was impaired early after HSCT, but restored at 24 months post-HSCT. MAIT cells have an increased sensibility to common immunosuppressive drugs, which maybe could explain their hampered reconstitution after HSCT.

Frontline Science: Placenta-derived decidual stromal cells alter IL-2R expression and signaling in alloantigen-activated T cells.

This study investigated how stromal cells affect the IL-2 pathway in alloantigen-activated T cells. We found that decidual stromal cells (DSCs) from term placentas promoted a high production of IL-2 in cultures with alloantigen-activated T cells. The intensity of expression of cluster of differentiation 25 (CD25; IL-2Rα) on T cells was increased by DSCs, whereas the frequency and intensity of expression of the signaling subunits CD122 (IL-2Rß) and CD132 (IL-2Rγc) were reduced. Consequently, uptake of IL-2 and STAT5 phosphorylation (pSTAT5) was abrogated. DSCs also decreased the proportion of pSTAT5+ T cells in response to IL-15, which also use CD122 for signaling. Addition of DSCs to the allogeneic cultures did not increase the expression of programmed death 1 (PD-1) or CD95, indicating that they did not promote T cell exhaustion. However, exogenous recombinant (r)IL-2 in similar concentrations in the same setting increased the expression of CD95 and down-regulated CD122 in T cells. The antiproliferative effect of sirolimus (SRL) and cyclosporine A (CsA), which target the IL-2 signaling pathway, was diminished by DSCs in vitro. To conclude, DSCs affect IL-2 production and IL-2R expression and signaling, which may contribute to the stromal cell-mediated immune modulation and phenotype shift seen in activated T cells. Altered proliferation in cultures when combining DSCs and SRL or CsA may be of clinical importance, as stromal cells are used in trials for acute inflammation and are often used in combination with conventional immunosuppressive therapies.

MAIT cells accumulate in placental intervillous space and display a highly cytotoxic phenotype upon bacterial stimulation.

During pregnancy, the maternal immune system must tolerate the developing foetus, and yet retain a potent antimicrobial response to prevent infections. Mucosal associated invariant T (MAIT) cells recognize microbial-derived vitamin B metabolites presented on the MR1 molecule, but their presence and function at the foetal-maternal interface is not known. We here isolated mononuclear cells from paired samples of peripheral blood (PB), intervillous blood (IVB), and decidua parietalis (DP) following uncomplicated term pregnancies. Interestingly, MAIT cells were highly enriched in IVB compared to PB and DP. The activation status of IVB MAIT cells was similar to that of PB MAIT cells, except for a lower expression of PD-1. Both IVB MAIT cells and conventional T cells were more dominated by an effector memory phenotype compared to PB MAIT cells and T cells. IVB MAIT cells also responded more vigorously with expression of IFN-γ, granzyme B, and perforin in response to Escherichia coli stimulation compared to PB. MR1 was not expressed in syncytiotrophoblasts, but in placental villous and decidual macrophages. These data indicate that maternal MAIT cells accumulate in the intervillous space of the placenta and that they are highly armed to quickly respond if bacteria are encountered at the foetal-maternal interface.

Who is the best hematopoietic stem-cell donor for a male patient with acute leukemia?

BACKGROUND: Female donors for male recipients worsen the outcome of allogeneic hematopoietic stem-cell transplantation. We wanted to find out whether a male human leukocyte antigen (HLA)-matched unrelated donor (MUD, 8/8, n=2,014) might be an alternative to a female HLA-identical sibling donor (n=2,656) for male patients with acute leukemia. METHODS: This is a retrospective analysis from the Acute Leukaemia Working Party of the European Group for Blood and Marrow Transplantation. RESULTS: The relative risk (RR) of acute graft-versus-host disease (GVHD) of grades II to IV was increased in the MUD group with acute myeloid leukemia (AML) (RR, 1.47; P<0.001) and acute lymphoblastic leukemia (ALL) (RR, 1.76; P<0.001). There was no difference in incidence of chronic GVHD and nonrelapse mortality between the two groups. Probability of relapse was lower in the MUD group than in the sibling group in patients with ALL (hazards ratio [HR], 0.75; P=0.04) but not in the AML patients (HR, 0.89; P=0.17). Survival was not different between the groups. Leukemia-free survival (LFS) was also similar in the sibling and MUD groups in patients with AML (HR, 1.01; P=0.81) or ALL (HR, 0.93; P=0.45). Factors significantly associated with reduced LFS included active disease, poor cytogenetics, age, year of hematopoietic stem-cell transplantation, reduced-intensity conditioning, and the use of antithymocyte globulin. CONCLUSION: Male patients who received grafts from male MUDs demonstrated an increased incidence of acute GVHD and LFS same as when using HLA-identical female donors.