Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice.

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

Epstein-Barr Virus Type 2 Infects T Cells and Induces B Cell Lymphomagenesis in Humanized Mice.

Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells in vitro suggest that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T cells, infecting T cells in culture and in healthy Kenyan infants, strongly suggesting that EBV-2 infection of T cells is a natural part of the EBV-2 life cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T cells. Therefore, BALB/c Rag2null IL2rγnull SIRPα humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T and B cells, unlike infection with EBV-1, in which only B cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B and T cells. Importantly, EBV-2-infected mice developed tumors resembling diffuse large B cell lymphoma (DLBCL). These lymphomas had morphological features comparable to those of EBV-1-induced DLBCLs, developed at similar rates with equivalent frequencies, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B cells in vitro, EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T cells, and the capacity of EBV-2 to drive lymphomagenesis.IMPORTANCE EBV is a well-established B cell-tropic virus. However, we have recently shown that the EBV type 2 (EBV-2) strain also infects primary T cells in culture and in healthy Kenyan children. This finding suggests that EBV-2, unlike the well-studied EBV-1 strain, utilizes the T cell compartment to persist. As EBV is human specific, studies to understand the role of T cells in EBV-2 persistence require an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34+ stem cells. Characterization of the EBV-2-infected humanized mice established that both T cells and B cells are infected by EBV-2 and that the majority of infected mice develop a B cell lymphoma resembling diffuse large B cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T cells contributes to persistence and lymphomagenesis.

Selecting the Best Donor for Haploidentical Transplant: Impact of HLA, Killer Cell Immunoglobulin-Like Receptor Genotyping, and Other Clinical Variables.

The use of post-transplant cyclophosphamide (PTCy)-based haploidentical (haplo) transplant is increasing worldwide. However, because multiple potential haplo donors are usually available, data-driven guidance is clearly needed to help transplant centers prioritize donors. To that end, we retrospectively analyzed 208 consecutive donor-recipient pairs receiving PTCy-based haplo transplant at a single institution. Median recipient and donor age were 52 years (range, 19 to 75) and 38 years (range, 15 to 73), peripheral blood stem cell was the stem cell source in 66%, and myeloablative conditioning was used in 41%. Median follow-up for surviving patients was 33 months (range, 7 to 130). Donor variables analyzed included age, sex, relationship, cytomegalovirus (CMV) status, ABO compatibility, HLA disparity, and several natural killer (NK) alloreactivity models. Multivariate Cox analysis was used to adjust for known patient, disease, and transplant covariates. Donor characteristics independently associated with improved survival included presence of HLA-DR mismatch, HLA-DP nonpermissive mismatch, killer cell immunoglobulin-like receptor (KIR) receptor-ligand mismatch, and KIR B/x haplotype with KIR2DS2. Donor characteristics associated with inferior survival included parental donor relationship and the use of a CMV-seronegative donor for a CMV-seropositive patient. Increased HLA disparity (≥4/10 HLA allelic mismatches [graft-versus-host direction]) resulted in relapse protection at the expense of increased nonrelapse mortality with no associated survival effect. We further propose a donor risk factor scoring system to permit a more evidence-based selection algorithm for potential haplo donors. This large, single-institution analysis demonstrates the importance of HLA-DR/HLA-DP disparity, NK alloreactivity, and other clinical variables in the haplo donor selection process and suggests that KIR and HLA-DP genotyping should be performed routinely for haplo donor selection.

Programmed Death 1 Expression on CD4+ T Cells Predicts Mortality after Allogeneic Stem Cell Transplantation.

Excessive or persistent programmed death 1 (PD-1) expression on virus- or tumor-specific T cells during chronic viral infection or malignancy has been associated with impaired immune control. To assess the role of the PD-1 pathway in allogeneic stem cell transplantation (SCT), we examined PD-1 expression and maturation phenotype on T cells from 42 patients early (day 55 to 85) after cord blood (CB), matched unrelated donor, and matched related donor transplantation. Expression of PD-1 on CD4+ T cells was significantly elevated in all transplantation types, with the highest level observed in CB subjects. Elevated PD-1 expression on CD4+ T cells early after transplantation was observed in nonsurvivors (median, 40.2%; range, 15.1 to 86.1) compared with survivors (median, 23.6%; range, 8.4 to 55.2; P = .001), indicating its association with increased risk for mortality, especially with CB transplantations, where PD-1 was increased in nonsurvivors (median, 64.6%; range, 36.5 to 86.1) compared with survivors (median, 34.1%; range, 15.9 to 55.2; P = .01). Furthermore, T cell subset analysis revealed that PD-1 expression was further elevated on CD4+ T central memory in nonsurvivors (median, 49.8%; range, 15.1 to 83.4) compared with survivors (median, 24.8%; range, 8.9 to 71.3; P = .002) and on T effector memory cells in nonsurvivors (median, 69.1%; range, 24.7 to 92.6) compared with survivors (median, 43.7%; range, 13.9 to 96.5; P = .0003). Our findings suggest that elevation of PD-1 expression on CD4+ T cells is associated with mortality in CB and possibly all SCT recipients.

Arthritogenic peptide binding to DRB1*01 alleles correlates with susceptibility to rheumatoid arthritis.

Genetic susceptibility to rheumatoid arthritis (RA) is often defined by the presence of a shared epitope (QKRAA, QRRAA, or RRRAA) at positions 70-74 in HLA-DRß1. However, DRß1*01:01 and 01:02 contain the same QRRAA epitope, but differ considerably in their susceptibility to RA. The purpose of this study was to determine if this difference could be explained by their ability to bind three arthritogenic peptides that we have previously shown to bind to the archetypal RA-susceptible allele, DRß1*04:01, but not to the resistant DRß1*08:01 allele. Binding of type II collagen(258-272), citrullinated and native vimentin(66-78), and citrullinated and native α-enolase(11-25) were measured on cell lines expressing either DRß1*01:01, *01:02 or *01:03 in association with DRα1*01:01. DRß1*01:01 and *01:02 both exhibited a 6.5-fold preference for citrullinated vimentin(66-78) compared to native vimentin. However, DRß1*01:01 also exhibited a 1.7-fold preference for citrullinated α-enolase(11-25) and bound collagen(258-272), while DRß1*01:02 bound neither of these peptides. Consistent with its known resistance to RA, DRß1*01:03 preferentially bound native vimentin(66-78) and α-enolase(11-25) over the citrullinated forms of these peptides, and also failed to bind collagen(258-272). Site-directed mutagenesis was performed to determine which amino acid residues were responsible for the differences between these alleles. Mutating position 86 in DRß1*01:01 from glycine to the valine residue found in DRß1*01:02 eliminated binding of both citrullinated α-enolase(11-25) and collagen(258-272), thereby recapitulating the peptide-binding profile of DRß1*01:02. The difference in susceptibility to rheumatoid arthritis between DRß1*01:01 and *01:02 thus correlates with the effect of position 86 on the binding of these arthritogenic peptides. Consistent with their association with RA resistance, positions I67, D70 and E71 all contributed to the inability of DRß1*01:03 to bind these arthritogenic peptides.

Studies of lymphocyte reconstitution in a humanized mouse model reveal a requirement of T cells for human B cell maturation.

The hematopoietic humanized mouse (hu-mouse) model is a powerful resource to study and manipulate the human immune system. However, a major and recurrent issue with this model has been the poor maturation of B cells that fail to progress beyond the transitional B cell stage. Of interest, a similar problem has been reported in transplant patients who receive cord blood stem cells. In this study, we characterize the development of human B and T cells in the lymph nodes (LNs) and spleen of BALB/c-Rag2(null)Il2rγ(null) hu-mice. We find a dominant population of immature B cells in the blood and spleen early, followed by a population of human T cells, coincident with the detection of LNs. Notably, in older mice we observe a major population of mature B cells in LNs and in the spleens of mice with higher T cell frequencies. Moreover, we demonstrate that T cells are necessary for B cell maturation, as introduction of autologous human T cells expedites the appearance of mature B cells, whereas in vivo depletion of T cells retards B cell maturation. The presence of the mature B cell population correlates with enhanced IgG and Ag-specific responses to both T cell-dependent and T cell-independent challenges, indicating their functionality. These findings enhance our understanding of human B cell development, provide increased details of the reconstitution dynamics of hu-mice, and validate the use of this animal model to study mechanisms and treatments for the similar delay of functional B cells associated with cord blood transplantations.

Results of the Phase 1 Open-Label Safety Study of Umbilical Cord Lining Mesenchymal Stromal/Stem Cells (Corlicyte®) to Heal Chronic Diabetic Foot Ulcers.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) play a critical role in wound healing. Corlicyte® is an MSC product derived from allogeneic umbilical cord tissue donated under an institutional review board-approved protocol and processed in accordance with section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act. This open-label phase 1 trial was performed under a United States Food and Drug Administration Investigational New Drug Application to establish the safety and tolerability of Corlicyte® in patients with diabetes and chronic diabetic foot ulcer (DFU). METHODS: Escalating doses were applied topically twice a week for up to 8 weeks after ulcer debridement, wound photography, and measurement. Subjects were followed for 4 weeks after the treatment phase. Adverse events were assessed at every visit. RESULTS: Nine subjects in 2 dosing cohorts completed the trial. No subjects experienced a serious adverse reaction to Corlicyte® or the development of anti-human leukocyte antigen (HLA) antibodies. Sixty percentage of subjects in the lower dose cohort experienced ulcer closure by Day 70 of follow-up, while the mean ulcer size was reduced by 54-67% in the other subjects. CONCLUSIONS: Topical administration of Corlicyte®, a novel biologic therapy consisting of allogeneic umbilical cord lining MSCs, appeared safe and tolerable and resulted in a significant decrease in ulcer area, demonstrating its potential as a therapy for healing of chronic DFU.

Ex vivo and in vivo evidence that cigarette smoke-exposed T regulatory cells impair host immunity against Mycobacterium tuberculosis.

Introduction: A strong epidemiologic link exists between cigarette smoke (CS) exposure and susceptibility to tuberculosis (TB). Macrophage and murine studies showed that CS and nicotine impair host-protective immune cells against Mycobacterium tuberculosis (MTB) infection. While CS and nicotine may activate T regulatory cells (Tregs), little is known about how CS may affect these immunosuppressive cells with MTB infection. Methods: We investigated whether CS-exposed Tregs could exacerbate MTB infection in co-culture with human macrophages and in recipient mice that underwent adoptive transfer of Tregs from donor CS-exposed mice. Results: We found that exposure of primary human Tregs to CS extract impaired the ability of unexposed human macrophages to control an MTB infection by inhibiting phagosome-lysosome fusion and autophagosome formation. Neutralizing CTLA-4 on the CS extract-exposed Tregs abrogated the impaired control of MTB infection in the macrophage and Treg co-cultures. In Foxp3+GFP+DTR+ (Thy1.2) mice depleted of endogenous Tregs, adoptive transfer of Tregs from donor CS-exposed B6.PL(Thy1.1) mice with subsequent MTB infection of the Thy1.2 mice resulted in a greater burden of MTB in the lungs and spleens than those that received Tregs from air-exposed mice. Mice that received Tregs from donor CS-exposed mice and infected with MTB had modest but significantly reduced numbers of interleukin-12-positive dendritic cells and interferon-gamma-positive CD4+ T cells in the lungs, and an increased number of total programmed cell death protein-1 (PD-1) positive CD4+ T cells in both the lungs and spleens. Discussion: Previous studies demonstrated that CS impairs macrophages and host-protective T effector cells in controlling MTB infection. We now show that CS-exposed Tregs can also impair control of MTB in co-culture with macrophages and in a murine model.

Unrelated donor cord blood transplantation for children with severe sickle cell disease: results of one cohort from the phase II study from the Blood and Marrow Transplant Clinical Trials Network (BMT CTN).

The Sickle Cell Unrelated Donor Transplant Trial (SCURT trial) of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) is a phase II study of the toxicity and efficacy of unrelated donor hematopoietic cell transplantation in children with severe sickle cell disease (SCD) using a reduced-intensity conditioning regimen. Here we report the results for the cord blood cohort of this trial. Eight children with severe SCD underwent unrelated donor cord blood transplantation (CBT) following alemtuzumab, fludarabine, and melphalan. Cyclosporine or tacrolimus and mycophenolate mofetil were administered for graft-versus-host disease (GVHD) prophylaxis. Donor/recipient HLA match status was 6 of 6 (n = 1) or 5 of 6 (n = 7), based on low/intermediate-resolution molecular typing at HLA -A, -B, and high-resolution typing at -DRB1. Median recipient age was 13.7 years (range: 7.4-16.2 years), and median weight was 35.0 kg (range: 25.2-90.2 kg). The median pre-cryopreservation total nucleated cell dose was 6.4 × 10(7) /kg (range: 3.1-7.6), and the median postthaw infused CD34 cell dose was 1.5 × 10(5) /kg (range: 0.2-2.3). All patients achieved neutrophil recovery (absolute neutrophil count >500/mm(3)) by day 33 (median: 22 days). Three patients who engrafted had 100% donor cells by day 100, which was sustained, and 5 patients had autologous hematopoietic recovery. Six of 8 patients had a platelet recovery to >50,000/mm(3) by day 100. Two patients developed grade II acute GVHD. Of these, 1 developed extensive chronic GVHD and died of respiratory failure 14 months posttransplantation. With a median follow-up of 1.8 years (range: 1-2.6), 7 patients are alive with a 1-year survival of 100%, and 3 of 8 are alive without graft failure or disease recurrence. Based upon the high incidence of graft rejection after unrelated donor CBT, enrollment onto the cord blood arm of the SCURT trial was suspended. However, because this reduced-intensity regimen has demonstrated a favorable safety profile, this trial remains open to enrollment for unrelated marrow donor transplants. Novel approaches aimed at improving engraftment will be needed before unrelated CBT can be widely adopted for transplanting patients with severe SCD.

Association of the HLA-DRB1 epitope LA(67, 74) with rheumatoid arthritis and citrullinated vimentin binding.

OBJECTIVE: Although rheumatoid arthritis (RA) has long been associated with an HLA-DRB1 shared epitope, a systematic search for other epitopes has never been conducted. In addition, the relationship between these epitopes and the binding of citrullinated autoantigens has not been investigated. We developed a program that can analyze HLA data for all possible epitopes of up to 5 amino acids and used this program to assess the shared epitope hypothesis in RA. METHODS: We analyzed high-resolution data from the International Histocompatibility Working Group, which included a group of 488 patients with RA and a group of 448 racially and ethnically balanced control subjects, for all combinations of up to 5 amino acids among polymorphic HLA-DRB1 positions 8-93. Statistical significance was determined by chi-square and Fisher's exact tests, with a false discovery rate correction. RESULTS: Three residues (V(11), H(13), and L(67)) were found to have the highest degree of association with RA susceptibility (P < 10(-11)), and D(70) was found to correlate best with RA resistance (P = 2 × 10(-11)). Of >2 million epitopes examined, LA(67, 74) exhibited the highest correlation with RA susceptibility (P = 2 × 10(-20); odds ratio 4.07 [95% confidence interval 3.07-5.39]). HLA alleles containing the LA(67, 74) epitope exhibited significantly greater binding to citrullinated vimentin(65-77) than did alleles containing D(70). Only 1 allele (DRB1*16:02) contained both LA(67, 74) and D(70); it bound citrullinated vimentin weakly and was not associated with RA. CONCLUSION: The findings of these studies confirm the importance of HLA-DRB1 amino acids in pocket 4 for the binding of citrullinated autoantigens and susceptibility to RA.

Cigarette smoke increases susceptibility to tuberculosis--evidence from in vivo and in vitro models.

BACKGROUND: Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. METHODS: We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. RESULTS: CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CONCLUSION: CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.

Lineage-Specific Relapse Prediction After Haploidentical Transplantation With Post-Transplant Cyclophosphamide Based on Recipient HLA-B-Leader Genotype and HLA-C-Group KIR Ligand.

The role of NK cell alloreactivity on outcomes after T cell-replete haploidentical donor transplantation (HIDT) remains uncertain. After transplantation, newly formed NK cells are licensed through interactions of donor inhibitory KIR (iKIR) and NKG2A receptors with their cognate ligands on recipient cells. Donor NKG2A recognizes HLA-E bound by recipient HLA class I leader peptides, a process requiring methionine (M) at position -21 of the leader sequence. An rs1050458C/T dimorphism results in approximately 40% of individuals expressing at least one copy of -21M HLA-B (M/M or M/T [M+]), allowing ligand expression. We assessed the impact of recipient HLA-B-leader genotype (M+ versus M- [T/T]) and HLA-C-group iKIR missing ligand (ML, C1C1/C2C2 versus C1C2) on relapse and disease-free survival (DFS) in recipients of post-transplantation cyclophosphamide (PTCy)-based HIDT. Based on preclinical data, we hypothesized that the relative impact of each variable may depend on disease lineage (lymphoid versus myeloid). To this end, we analyzed outcomes of 322 consecutive PTCy-based HIDT recipients with hematologic malignancy who underwent transplantation at a single institution using standardized supportive care measures with mature follow-up (median 45 months). Primary endpoints were relapse and DFS of patients based on HLA-B-leader genotype and HLA-C-group iKIR ML. Planned subgroup analysis included patient with lymphoid versus myeloid malignancy. M+ HLA-B-leader genotype and HLA-C-group iKIR ML were seen in 42% and 49% of recipients, respectively. The presence of a recipient M+ B-leader (versus M-) improved overall survival (OS) and DFS and lowered cumulative incidence of relapse (CIR), an effect primarily seen in lymphoid malignancies (80% versus 51%, 72% versus 41%, 16% versus 42%, respectively). In contrast, myeloid malignancy patients benefited most from HLA-C-group iKIR ML with better OS and DFS and lower CIR (67% versus 51%, 64% versus 44%, 25% versus 45%, respectively). Multivariate analysis confirmed the disease-specific associations of improved relapse/DFS with M+ HLA-B-leader in lymphoid malignancy (hazard ratio [HR] 0.20, P < .001/HR 0.34, P <.001) and HLA-C-group iKIR ML in myeloid malignancy (HR 0.44, P = .004/HR 0.54, P = .009). Neither HLA-B-leader nor iKIR ML was associated with the incidence of non-relapse mortality or acute or chronic graft-versus-host disease. Two distinct NK cell education pathways predict relapse and DFS after HIDT-PTCy in a disease-specific manner: the presence of recipient M+ HLA-B-leader genotype improves outcome in patients with lymphoid malignancies, whereas HLA-C-group iKIR ML improves outcome in patients with myeloid malignancies. These findings strengthen the essential role of NK cells for optimal GVL in the context of HIDT-PTCy and may suggest different approaches to improving transplant outcome depending on disease type.

HLA homozygosity is associated with Non-Hodgkin lymphoma.

The "heterozygote advantage" hypothesis has been postulated regarding the role of human leukocyte antigen (HLA) in non-Hodgkin lymphoma (NHL), where homozygous loci are associated with an increased risk of disease. In this retrospective study, we analyzed the HLA homozygosity of 3789 patients with aplastic anemia (AA), acute lymphocytic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) at HLA-A, B, C, DRB1 and DQB1 loci compared to 169,964 normal controls. HLA homozygosity at one or more loci was only associated with an increased risk in NHL patients (OR = 1.28, 95% CI [1.09, 1.50], p = 0.002). This association was not seen in any of the other hematologic diseases. Homozygosity at HLA-A alone, HLA-B + C only, and HLA-DRB1 + DQB1 only was also significantly associated with NHL. Finally, we observed a 17% increased risk of NHL with each additional homozygous locus (OR per locus = 1.17, 95% CI [1.08, 1.25], p trend = 2.4 × 10-5). These results suggest that reduction of HLA diversity could predispose individuals to an increased risk of developing NHL.

Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19.

The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. We determined whether negatively-charged heparin enhanced TMPRSS2 inhibition by alpha-1-antitrypsin (AAT). TMPRSS2 activity was determined in HEK293T cells overexpressing TMPRSS2. We quantified infection of primary human airway epithelial cells (hAEc) with human coronavirus 229E (HCoV-229E) by immunostaining for the nucleocapsid protein and by the plaque assay. Detailed molecular modeling was undertaken with the heparin-TMPRSS2-AAT ternary complex. Enoxaparin enhanced AAT inhibition of both TMPRSS2 activity and infection of hAEc with HCoV-229E. Underlying these findings, detailed molecular modeling revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2-AAT interface, neutralizing otherwise repulsive forces. In conclusion, enoxaparin enhances AAT inhibition of both TMPRSS2 and coronavirus infection. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.

Cabozantinib sensitizes microsatellite stable colorectal cancer to immune checkpoint blockade by immune modulation in human immune system mouse models.

Immune checkpoint inhibitors have been found to be effective in metastatic MSI-high colorectal cancers (CRC), however, have no efficacy in microsatellite stable (MSS) cancers, which comprise the majority of mCRC cases. Cabozantinib is a small molecule multi-tyrosine kinase inhibitor that is FDA approved in advanced renal cell, medullary thyroid, and hepatocellular carcinoma. Using Human Immune System (HIS) mice, we tested the ability of cabozantinib to prime MSS-CRC tumors to enhance the potency of immune checkpoint inhibitor nivolumab. In four independent experiments, we implanted distinct MSS-CRC patient-derived xenografts (PDXs) into the flanks of humanized BALB/c-Rag2nullIl2rγnullSirpαNOD (BRGS) mice that had been engrafted with human hematopoietic stem cells at birth. For each PDX, HIS-mice cohorts were treated with vehicle, nivolumab, cabozantinib, or the combination. In three out of the four models, the combination had a lower tumor growth rate compared to vehicle or nivolumab-treated groups. Furthermore, interrogation of the HIS in immune organs and tumors by flow cytometry revealed increased Granzyme B+, TNFα+ and IFNγ+ CD4+ T cells among the human tumor infiltrating leukocytes (TIL) that correlated with reduced tumor growth in the combination-treated HIS-mice. Notably, slower growth correlated with increased expression of the CD4+ T cell ligand, HLA-DR, on the tumor cells themselves. Finally, the cabozantinib/nivolumab combination was tested in comparison to cobimetinib/atezolizumab. Although both combinations showed tumor growth inhibition, cabozantinib/nivolumab had enhanced cytotoxic IFNγ and TNFα+ T cells. This pre-clinical in vivo data warrants testing the combination in clinical trials for patients with MSS-CRC.

Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rγnull mouse model: a multivariable optimization approach.

Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2(null)Il2rγ(null) strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34(-) cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2(null)Il2rγ(null) humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.

Hypothesis: Alpha-1-antitrypsin is a promising treatment option for COVID-19.

No definitive treatment for COVID-19 exists although promising results have been reported with remdesivir and glucocorticoids. Short of a truly effective preventive or curative vaccine against SARS-CoV-2, it is becoming increasingly clear that multiple pathophysiologic processes seen with COVID-19 as well as SARS-CoV-2 itself should be targeted. Because alpha-1-antitrypsin (AAT) embraces a panoply of biologic activities that may antagonize several pathophysiologic mechanisms induced by SARS-CoV-2, we hypothesize that this naturally occurring molecule is a promising agent to ameliorate COVID-19. We posit at least seven different mechanisms by which AAT may alleviate COVID-19. First, AAT is a serine protease inhibitor (SERPIN) shown to inhibit TMPRSS-2, the host serine protease that cleaves the spike protein of SARS-CoV-2, a necessary preparatory step for the virus to bind its cell surface receptor ACE2 to gain intracellular entry. Second, AAT has anti-viral activity against other RNA viruses HIV and influenza as well as induces autophagy, a known host effector mechanism against MERS-CoV, a related coronavirus that causes the Middle East Respiratory Syndrome. Third, AAT has potent anti-inflammatory properties, in part through inhibiting both nuclear factor-kappa B (NFκB) activation and ADAM17 (also known as tumor necrosis factor-alpha converting enzyme), and thus may dampen the hyper-inflammatory response of COVID-19. Fourth, AAT inhibits neutrophil elastase, a serine protease that helps recruit potentially injurious neutrophils and implicated in acute lung injury. AAT inhibition of ADAM17 also prevents shedding of ACE2 and hence may preserve ACE2 inhibition of bradykinin, reducing the ability of bradykinin to cause a capillary leak in COVID-19. Fifth, AAT inhibits thrombin, and venous thromboembolism and in situ microthrombi and macrothrombi are increasingly implicated in COVID-19. Sixth, AAT inhibition of elastase can antagonize the formation of neutrophil extracellular traps (NETs), a complex extracellular structure comprised of neutrophil-derived DNA, histones, and proteases, and implicated in the immunothrombosis of COVID-19; indeed, AAT has been shown to change the shape and adherence of non-COVID-19-related NETs. Seventh, AAT inhibition of endothelial cell apoptosis may limit the endothelial injury linked to severe COVID-19-associated acute lung injury, multi-organ dysfunction, and pre-eclampsia-like syndrome seen in gravid women. Furthermore, because both NETs formation and the presence of anti-phospholipid antibodies are increased in both COVID-19 and non-COVID pre-eclampsia, it suggests a similar vascular pathogenesis in both disorders. As a final point, AAT has an excellent safety profile when administered to patients with AAT deficiency and is dosed intravenously once weekly but also comes in an inhaled preparation. Thus, AAT is an appealing drug candidate to treat COVID-19 and should be studied.

Testing Cancer Immunotherapy in a Human Immune System Mouse Model: Correlating Treatment Responses to Human Chimerism, Therapeutic Variables and Immune Cell Phenotypes.

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of "responding" patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis.

Peanut-allergic subjects and their peanut-tolerant siblings have large differences in peanut-specific IgG that are independent of HLA class II.

We enrolled 53 peanut-allergic subjects and 64 peanut-tolerant full siblings, measured peanut-specific IgG and IgE, determined HLA class II at high resolution, and analyzed DRB1 alleles by supertypes. Peanut-specific IgG and IgE were elevated in the peanut-allergic subjects (p<0.0001) but did not stratify with HLA alleles, haplotypes, or supertypes. There were no significant differences in HLA class II between the peanut-allergic and peanut-tolerant siblings but there was an increased frequency of DRB1*0803 in both sets of siblings compared to unrelated controls (p(c)=4.5×10⁻9). Furthermore, we identified 14 sibling pairs in which the peanut-allergic and the peanut-tolerant siblings have identical HLA class II and again found an elevation of anti-peanut IgG in the peanut-allergic subjects (p<0.0001). In conclusion, although DRB1*0803 may identify a subset of families with increased risk of peanut allergy, differences in peanut-specific immunoglobulin production between peanut-allergic subjects and their peanut-tolerant siblings are independent of HLA class II.