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.

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.

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.

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.

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.

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.

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.

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.

Development of an Adrenocortical Cancer Humanized Mouse Model to Characterize Anti-PD1 Effects on Tumor Microenvironment.

CONTEXT: Although the development of immune checkpoint inhibitors has transformed treatment strategies of several human malignancies, research models to study immunotherapy in adrenocortical carcinoma (ACC) are lacking. OBJECTIVE: To explore the effect of anti-PD1 immunotherapy on the alteration of the immune milieu in ACC in a newly generated preclinical model and correlate with the response of the matched patient. DESIGN, SETTING, AND INTERVENTION: To characterize the CU-ACC2-M2B patient-derived xenograft in a humanized mouse model, evaluate the effect of a PD-1 inhibitor therapy, and compare it with the CU-ACC2 patient with metastatic disease. RESULTS: Characterization of the CU-ACC2-humanized cord blood-BALB/c-Rag2nullIl2rγnullSirpaNOD model confirmed ACC origin and match with the original human tumor. Treatment of the mice with pembrolizumab demonstrated significant tumor growth inhibition (60%) compared with controls, which correlated with increased tumor infiltrating lymphocyte activity, with an increase of human CD8+ T cells (P < 0.05), HLA-DR+ T cells (P < 0.05) as well as Granzyme B+ CD8+ T cells (<0.001). In parallel, treatment of the CU-ACC2 patient, who had progressive disease, demonstrated a partial response with 79% to 100% reduction in the size of target lesions, and no new sites of metastasis. Pretreatment analysis of the patient's metastatic liver lesion demonstrated abundant intratumoral CD8+ T cells by immunohistochemistry. CONCLUSIONS: Our study reports the first humanized ACC patient-derived xenograft mouse model, which may be useful to define mechanisms and biomarkers of response and resistance to immune-based therapies, to ultimately provide more personalized care for patients with ACC.

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.

Replacing mouse BAFF with human BAFF does not improve B-cell maturation in hematopoietic humanized mice.

Hematopoietic humanized mice (hu-mice) have been developed to study the human immune system in an experimental in vivo model, and experiments to improve its performance are ongoing. Previous studies have suggested that the impaired maturation of human B cells observed in hu-mice might be in part due to inefficient interaction of the human B-cell-activating factor (hBAFF) receptor with mouse B-cell-activating factor (mBAFF), as this cytokine is an important homeostatic and differentiation factor for B lymphocytes both in mice and humans. To investigate this hypothesis, we created a genetically engineered mouse strain in which a complementary DNA (cDNA) encoding full-length hBAFF replaces the mBAFF-encoding gene. Expression of hBAFF in the endogenous mouse locus did not lead to higher numbers of mature and effector human B cells in hu-mice. Instead, B cells from hBAFF knock-in (hBAFFKI) hu-mice were in proportion more immature than those of hu-mice expressing mBAFF. Memory B cells, plasmablasts, and plasma cells were also significantly reduced, a phenotype that associated with diminished levels of immunoglobulin G and T-cell-independent antibody responses. Although the reasons for these findings are still unclear, our data suggest that the inefficient B-cell maturation in hu-mice is not due to suboptimal bioactivity of mBAFF on human B cells.

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.

A Molecular Analysis of the Shared Epitope Hypothesis: Binding of Arthritogenic Peptides to DRB1*04 Alleles.

OBJECTIVE: The shared epitope hypothesis posits that amino acids QR/KRAA in positions 70-74 of the DRΒ1 chain are responsible for rheumatoid arthritis susceptibility. However, even DRB1*04 alleles containing the shared epitope vary greatly with respect to degrees of susceptibility. This study was undertaken to conduct a molecular examination of the shared epitope hypothesis by measuring binding of arthritogenic peptides to susceptibility and resistance alleles. METHODS: We measured binding of native and citrullinated forms of vimentin(66-78) and α-enolase(11-25) and noncitrullinated type II collagen(258-272) to 88 class II alleles on Luminex beads (which includes alleles of many varying degrees of susceptibility and resistance). We expressed DRΒ1*04:01, *04:02, and *08:01 in T2 cells and mutated DRΒ1*04:01 at positions 67, 70, 71, 74, and 86 to corresponding residues in DRB1*04:02, *04:03, *04:04, *04:05, and *08:01. Finally, we measured responses of 4 DRΒ1*04:01 restricted collagen(258-272) T cell hybridomas against wild-type DRΒ1*04:01, *04:02, and all mutated alleles. RESULTS: The most susceptible allele, DRΒ1*04:01, preferentially bound citrullinated vimentin(66-78) and citrullinated α-enolase(11-25) over the native forms. DRΒ1*04:02 exhibited no preference for citrullinated peptides, and *08:01 preferred native peptides. Similarly, DRB1*04:01 bound collagen(258-272) , but *04:02 and *08:01 did not. Mutating DRΒ1*04:01 at positions 70, 71, 74, and 86 to the corresponding residues in DRΒ1*04:02 or *08:01 dramatically reduced the specificity for citrullinated peptides and collagen(258-272) binding. CONCLUSION: These observations demonstrate that while amino acids at positions 70, 71, and 74 within the shared epitope in DRΒ1 mediate binding and T cell responses of arthritogenic peptides, position 86 outside the shared epitope also plays a critical role.

Receptor editing and genetic variability in human autoreactive B cells.

The mechanisms by which B cells undergo tolerance, such as receptor editing, clonal deletion, and anergy, have been established in mice. However, corroborating these mechanisms in humans remains challenging. To study how autoreactive human B cells undergo tolerance, we developed a novel humanized mouse model. Mice expressing an anti-human Igκ membrane protein to serve as a ubiquitous neo self-antigen (Ag) were transplanted with a human immune system. By following the fate of self-reactive human κ(+) B cells relative to nonautoreactive λ(+) cells, we show that tolerance of human B cells occurs at the first site of self-Ag encounter, the bone marrow, via a combination of receptor editing and clonal deletion. Moreover, the amount of available self-Ag and the genetics of the cord blood donor dictate the levels of central tolerance and autoreactive B cells in the periphery. Thus, this model can be useful for studying specific mechanisms of human B cell tolerance and to reveal differences in the extent of this process among human populations.

Detection of immunoglobulin isotypes from dried blood spots.

The study was designed to determine the sensitivity and reproducibility of recovering immunoglobulin (Ig) isotypes (IgG subclasses, IgA, IgE and IgM classes) from dried blood spots (DBS), a methodologic subcomponent of the Upstate KIDS Study. A multiplexed Luminex assay was used for IgG1/2/3/4, IgA and IgM analysis; an ELISA was used for IgE. Plasma samples from de-identified patients were used to compare the Luminex assay with nephelometry, which is routinely used to quantify IgA, IgG and IgM in clinical samples. The IgE ELISA was compared to an immunofluorescence assay. Prior to evaluation of punches from newborn dried blood spots (NDBSs), recoveries of Ig from punches of cord blood DBSs (CBDBSs) vs. plasma from the same cord bloods were compared. Although the recoveries of Ig from plasma and DBSs were not comparable, which could be due to cell lysates in the DBS samples, the analyses were reproducible. Additionally, the levels of IgA, IgG2, IgG4, and IgM recovered from CBDBSs positively correlated with those in plasma. The DBS data is a relative value since it is not equivalent to the plasma concentration. The majority of Ig concentrations recovered from 108 newborns of the Upstate KIDs Study were within the range of newborn plasma Ig levels with the exception of IgG3. The IgG4 values displayed the greatest variance with a wide range (0.01-319 mg/dl), whereas, IgG1 values had the narrowest range (85.2-960.4 mg/dl).