Murine pre-B-cell ALL induces T-cell dysfunction not fully reversed by introduction of a chimeric antigen receptor.

Adoptive transfer of patient-derived T cells modified to express chimeric antigen receptors (CARTs) has demonstrated dramatic success in relapsed/refractory pre-B-cell acute lymphoblastic leukemia (ALL), but response and durability of remission requires exponential CART expansion and persistence. Tumors are known to affect T-cell function, but this has not been well studied in ALL and in the context of chimeric antigen receptor (CAR) expression. Using TCF3/PBX1 and MLL-AF4-driven murine ALL models, we assessed the impact of progressive ALL on T-cell function in vivo. Vaccines protect against TCF3/PBX1.3 but were ineffective when administered after leukemia injection, suggesting immunosuppression induced early during ALL progression. T cells from leukemia-bearing mice exhibited increased expression of inhibitory receptors, including PD1, Tim3, and LAG3, and were dysfunctional following adoptive transfer in a model of T-cell receptor (TCR)-dependent leukemia clearance. Although expression of inhibitory receptors has been linked to TCR signaling, pre-B-cell ALL induced inhibitory receptor expression, at least in part, in a TCR-independent manner. Finally, introduction of a CAR into T cells generated from leukemia-bearing mice failed to fully reverse poor in vivo function.

Comparing patient outcomes between multiple ipsilateral iliac artery stents and isolated iliac artery stents.

OBJECTIVE: Endovascular stents are accepted therapy for iliac artery stenoses and occlusions. Surgery is the recommended therapy for patients with severe iliac artery disease, including those with the combination of ipsilateral common iliac artery (CIA) and external iliac artery (EIA) stenoses/occlusions. This study compared patient outcomes, including late open conversion rates, for combined ipsilateral CIA and EIA stenting vs CIA or EIA stents alone. METHODS: Between 1998 and 2010, 588 patients underwent iliac artery stenting at two institutions. Patient comorbidities and outcomes were retrospectively reviewed, and analyses were performed using multivariate regression and Kaplan-Meier methods. RESULTS: There were 436 extremities with CIA stents, 195 with EIA stents, and 157 with CIA and EIA stents. The groups did not differ significantly in demographics, comorbidities, or treatment indications. During follow-up, 183 patients died, 95 underwent an endovascular reintervention, and 48 required late open conversion. For patients in the CIA or EIA stent group, the mean ± standard error survival was 5.3 ± 0.3 years, secondary endovascular intervention-free survival was 7.4 ± 0.6 years, late open conversion-free survival was 9.8 ± 0.4 years, and amputation-free survival was 7.6 ± 0.4 years. In the CIA and EIA stent group, survival was 6.1 ± 0.6 years, secondary endovascular intervention-free survival was 7.2 ± 0.6 years, late open conversion-free survival was 9.0 ± 1.1 years, and amputation-free survival was 8.4 ± 0.5 years. Survival, reintervention-free survival, late open conversion-free survival, and amputation-free survival were all similar between patient groups (all P > .05). CIA and EIA stenting in combination was not a predictor of death, reintervention, late open conversion, or amputation. CONCLUSIONS: Outcomes are similar for patients with CIA or EIA stents and for those with combined ipsilateral CIA and EIA stents. Late open conversions for iliac artery stent failure are uncommon and not influenced by the location or extent of prior iliac artery stent placement. Endovascular therapy for aortoiliac disease should be extended to consider selected patients with ipsilateral CIA and EIA stenoses/occlusions.

Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens.

The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized.

Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens.

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.

Abs are central to malaria immunity, which is only acquired after years of exposure to Plasmodium falciparum (Pf). Despite the enormous worldwide burden of malaria, the targets of protective Abs and the basis of their inefficient acquisition are unknown. Addressing these knowledge gaps could accelerate malaria vaccine development. To this end, we developed a protein microarray containing approximately 23% of the Pf 5,400-protein proteome and used this array to probe plasma from 220 individuals between the ages of 2-10 years and 18-25 years in Mali before and after the 6-month malaria season. Episodes of malaria were detected by passive surveillance over the 8-month study period. Ab reactivity to Pf proteins rose dramatically in children during the malaria season; however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in 8-10-year-old children who were infected with Pf during the malaria season but did not experience malaria (n = 12) vs. those who experienced malaria (n = 29). This analysis also provided insight into patterns of Ab reactivity against Pf proteins based on the life cycle stage at which proteins are expressed, subcellular location, and other proteomic features. This approach, if validated in larger studies and in other epidemiological settings, could prove to be a useful strategy for better understanding fundamental properties of the human immune response to Pf and for identifying previously undescribed vaccine targets.

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens.

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.