Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform

An HLA-A*11:01-binding neoantigen from mutated NPM1 as target for TCR gene therapy in AML

Simple Summary: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis. For AML relapses after chemotherapy, new and effective therapies are needed. In 30-35% of AMLs, a frameshift mutation in the nucleophosmin 1 gene (dNPM1) creates potential neoantigens that are attractive targets for immunotherapy. We previously isolated a T-cell receptor (TCR) that targets an HLA-A*02:01-binding dNPM1 neoantigen on primary AML. Here, we investigated whether AVEEVSLRK is another dNPM1 neoantigen that can be targeted by TCR gene transfer. We isolated various T-cells, cloned the HLA-A*11:01-restricted TCR from one T-cell clone and, upon transfer to CD8 cells, demonstrated targeting of dNPM1 primary AMLs in vitro. However, the TCR failed to mediate an anti-tumor effect in immunodeficient mice engrafted with dNPM1 OCI-AML3 cells. Our results demonstrate that AVEEVSLRK is an HLA-A*11:01-binding neoantigen on dNPM1 AML. Whether the isolated TCR is of sufficient affinity to treat patients remains uncertain.Acute myeloid leukemia (AML) is a hematological malignancy caused by clonal expansion of myeloid progenitor cells. Most patients with AML respond to chemotherapy, but relapses often occur and infer a very poor prognosis. Thirty to thirty-five percent of AMLs carry a four base pair insertion in the nucleophosmin 1 gene (NPM1) with a C-terminal alternative reading frame of 11 amino acids. We previously identified various neopeptides from the alternative reading frame of mutant NPM1 (dNPM1) on primary AML and isolated an HLA-A*02:01-restricted T-cell receptor (TCR) that enables human T-cells to kill AML cells upon retroviral gene transfer. Here, we isolated T-cells recognizing the dNPM1 peptide AVEEVSLRK presented in HLA-A*11:01. The TCR cloned from a T-cell clone recognizing HLA-A*11:01+ primary AML cells conferred in vitro recognition and lysis of AML upon transfer to CD8 cells, but failed to induce an anti-tumor effect in immunodeficient NSG mice engrafted with dNPM1 OCI-AML3 cells. In conclusion, our data show that AVEEVSLRK is a dNPM1 neoantigen on HLA-A*11:01+ primary AMLs. CD8 cells transduced with an HLA-A*11:01-restricted TCR for dNPM1 were reactive against AML in vitro. The absence of reactivity in a preclinical mouse model requires further preclinical testing to predict the potential efficacy of this TCR in clinical development.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Famine food of vegetal origin consumed in the Netherlands during World War II

Background: Periods of extreme food shortages during war force people to eat food that they normally do not consider edible. The last time that countries in Western Europe experienced severe scarcities was during World War II. The so-called Dutch famine or Hunger Winter (1944-1945) made at least 25,000 victims. The Dutch government took action by opening soup kitchens and providing information on wild plants and other famine food sources in "wartime cookbooks." The Dutch wartime diet has never been examined from an ethnobotanical perspective. Methods: We interviewed 78 elderly Dutch citizens to verify what they remembered of the consumption of vegetal and fungal famine food during World War II by them and their close surroundings. We asked whether they experienced any adverse effects from consuming famine food plants and how they knew they were edible. We identified plant species mentioned during interviews by their local Dutch names and illustrated field guides and floras. We hypothesized that people living in rural areas consumed more wild species than urban people. A Welch t test was performed to verify whether the number of wild and cultivated species differed between urban and rural citizens. Results: A total number of 38 emergency food species (14 cultivated and 21 wild plants, three wild fungi) were mentioned during interviews. Sugar beets, tulip bulbs, and potato peels were most frequently consumed. Regularly eaten wild species were common nettle, blackberry, and beechnuts. Almost one third of our interviewees explicitly described to have experienced extreme hunger during the war. People from rural areas listed significantly more wild species than urban people. The number of cultivated species consumed by both groups was similar. Negative effects were limited to sore throats and stomachache from the consumption of sugar beets and tulip bulbs. Knowledge on the edibility of famine food was obtained largely by oral transmission; few people remembered the written recipes in wartime cookbooks. Conclusion: This research shows that 71years after the Second World War, knowledge on famine food species, once crucial for people's survival, is still present in the Dutch society. The information on famine food sources supplied by several institutions was not distributed widely. For the necessary revival of famine food knowledge during the 1940s, people needed to consult a small group of elders. Presumed toxicity was a major reason given by our participants to explain why they did not collect wild plants or mushrooms during the war

Impact of flowering phenology of Silene alba and S-dioica on susceptibility to fungal infection and seed predation

Timing of the production of susceptible host stages can have a large impact on a plant's probability of infection by pathogens and on the extent of damage caused by herbivores. In the closely related plant species Silene alba and S. dioica, flowers are the site of infection by the host-sterilizing anther smut fungus Ustilago violacea, as well as the site of oviposition by the noctuid Hadena bicruris. In diseased populations of the two host species, the noctuid can act as pollinator, fruit predator, and vector of fungal spores. Hence, host traits that enhance or reduce visitation rates by the noctuid could affect fruit set, fruit predation and infection probability. A field study was conducted to investigate the magnitude and direction of effects of the timing of anthesis on reproductive success via these pollinator-mediated processes. More than 50% of the Silene fruits produced in a sympatric population of S. alba and S. dioica were predated by the noctuid. Predation was two times higher in S. alba than in S. dioica, annihilating the two times higher flower and fruit production by the former species. This finding emphasizes the potential impact of biotic factors on the relative fecundity of related plant species in natural populations. Onset of flowering was 4-8 weeks earlier in S. dioica than in S. alba. Tn S. dioica, early flowering individuals produced the largest fraction of undamaged fruits per flower because of their high fruit set, and their low rates of fruit abortion and predation. By contrast, in S. alba, late flowering individuals produced the highest fraction of undamaged Fruits per flower, because of a monotonic decrease in Fruit predation with time and the absence of a significant impact of phenology on fruit set. In principle, pollinator-mediated selection could thus favour earlier flowering plants in S. dioica and later flowering plants in S. alba, and an evolutionary response to such selection would increase the average difference in onset of flowering between the two species. However, differences in fitness among phenology classes were more strongly determined by differences in flower production per plant than by differences in the average reproductive success per flower. A response to pollinator-mediated selection is therefore only expected if effects of age, size, or other factors that determine differences in flower production per plant are averaged out over progeny. In both host species, ca 20% of the hosts only produced sterile flowers as a result of systemic infection by the fungal pathogen U. violacea. In S. alba, the impact of phenology on fungal infection was in the same direction as for fruit predation (decreasing with later onset of flowering). In S. dioica, phenology had no effect on the probability of infection. In both species, associations between infection and predation tended to be positive but were not statistically significant, suggesting that the presence of alternative vectors or variation in resistance unrelated to traits affecting vector contact map prevent a tight association between damage caused by a vector-herbivore and its vectored disease. [KEYWORDS: Anther-smut infection; ustilago-violacea;spore deposition experimental populations; disease transmission; genotypic variation; life-history; patterns; caryophyllaceae; epidemiology]

Anther smut transmission in Silene latifolia and Silene dioica: Impact of host traits, disease frequency, and host density

Predicting the dynamics of hosts and pathogens in natural systems requires a thorough understanding of the disease transmission process. It has been argued that, unlike for airborne diseases, the transmission of vector-borne diseases such as the anther smut fungus Microbotryum violaceum is a function of the frequency rather than the density of disease in a population. However, recent models indicate that frequency- dependent transmission of vectored diseases is only expected if the time required for interplant movement (host search) by the vector is short relative to the time spent per visit (handling time). Hence, frequency dependence is only expected to dominate at lower ranges of interplant distances. We test this prediction by analyzing disease transmission of M. violaceum in relation to interplant distance in a natural population of the host species Silene latifolia and Silene dioica. We conclude that (1) The infection probability of uninfected hosts strongly increased with the frequency of diseased hosts within a patch, whereas the number of infected plants within a patch had no effect in either of the host species. (2) In agreement with predictions based on vector behavior, the probability of infection in patches of S. dioica increased more strongly with increasing disease frequency among patches with high average host density (i.e., small interplant distance) than among patches with low host density. This finding is consistent with the idea that transmission should shift from a more frequency- to a more density-dependent process when host search time increases relative to handling time. (3) In addition to local disease frequency and host density, individual host traits (host sex, size, and phenology) explained a significant part of the variation in infection probability within host species. The precise effects of individual host traits depended on local neighborhood characteristics. For instance, in S. latifolia, male hosts were on average more susceptible than female hosts, but the difference diminished with increasing disease frequency. [KEYWORDS: Violacea microbotryum-violaceum; ustilago-violacea; spore deposition; experimental populations; alba; infection; patterns; dynamics; epidemiology; genetics]

Host adaptation in the anther smut fungus Ustilago violacea (Microbotryum violaceum): Infection success, spore production and alteration of floral traits on two host species and their F1-hybrid

It is often assumed that host specialization is promoted by trade-offs in the performance of parasites on different host species, but experimental evidence for such trade-offs is scant. We studied differences in performance among strains of the anther smut fungus Ustilago violacea from two closely related host plant species, Silene alba and S. dioica, on progeny of (1) the host species from which they originated, (2) the alternative host species, and (3) inter-specific hybrids. Significant intra-specific variation in the pathogen was found for both infection success on a range of host genotypes (virulence) and components of spore production per infected host (aggressiveness) (sensu Burden 1987). Strains did not have overall higher virulence on conspecifics of their host of origin than on strains from the heterospecific host, but they did have a significantly (c. 3 times) higher spore production per infected male host. This finding suggests that host adaptation may have evolved with respect to aggressiveness rather than virulence. The higher aggressiveness of strains on conspecifics of their host of origin resulted both from higher spore production per infected flower (spores are produced in the anthers), and greater ability to stimulate flower production on infected hosts. The latter indicates the presence of adaptive intraspecific variation in the ability of host manipulation. As transmission of the fungus is mediated by insects that are both pollinators of the host and vectors of the disease, we also assessed the effect of strains on host floral traits. Infection resulted in a reduction of inflorescence height, flower size, and nectar production per flower. Strains did not differ in their effect on nectar production, but infection with strains from S. alba resulted in a stronger reduction of inflorescence height and petal size on both host species. Vectors may therefore in principle discriminate among hosts infected by different strains and affect their efficiency of transmission. Contrary to assumptions of recent hypotheses about the role of host hybrids in the evolution of parasites, hybrids were not generally more susceptible than parental hosts. It is therefore unlikely that the rate of evolution of the pathogen on the parental species is slowed down by selection for specialization on the hybrids. [KEYWORDS: host manipulation; host specialization; plant-pathogen interactions; Silene; Ustilago violacea Silene-alba; linum-marginale; experimental populations; pollinator visitation; pathogen interactions; natural-populations; genotypic variation; viscaria-vulgaris; melampsora-lini; disease spread]

Coping with third parties in a nursery pollination mutualism: Hadena bicruris avoids oviposition on pathogen-infected, less rewarding Silene latifolia

In nursery pollination systems, pollinator offspring usually feed on pollinated fruits or seeds. Costs and benefits of the interaction for plant and pollinator, and hence its local outcome (antagonismmutualism), can be affected by the presence of 'third-party' species. Infection of Silene latifolia plants by the fungus Microbotryum violaceum halts the development of fruits that provide shelter and food for larvae of the pollinating moth Hadena bicruris. We investigated whether the moth secures its benefit by selective oviposition on uninfected flowers. Oviposition was recorded in eight natural populations as a function of plant infection status, local neighbourhood, plant and flower characteristics. Oviposition was six times lower on flowers from infected than on those from uninfected plants. Oviposition decreased with decreasing flower and ovary size. Moths could use the latter to discriminate against diseased flowers. Although moths show an adaptive oviposition response, they reduce the future potential of healthy hosts because they still visit infected plants for nectar, vectoring the disease, and they reduce any fitness advantage gained by disease-resistant plants through selective predation of those plants. [KEYWORDS: Hadena bicruris ; Microbotryum violaceum ; mutualism ; nursery pollination ; oviposition ; plant pathogen ; seed predation ; Silene latifolia]