Transcriptome and unique cytokine microenvironment of Castleman disease.

Castleman disease (CD) represents a group of rare, heterogeneous and poorly understood disorders that share characteristic histopathological features. Unicentric CD (UCD) typically involves a single enlarged lymph node whereas multicentric CD (MCD) involves multiple lymph node stations. To understand the cellular basis of CD, we undertook a multi-platform analysis using targeted RNA sequencing, RNA in-situ hybridization (ISH), and adaptive immune receptor rearrangements (AIRR) profiling of archived tissue from 26 UCD, 14 MCD, and 31 non-CD reactive controls. UCD showed differential expression and upregulation of follicular dendritic cell markers (CXCL13, clusterin), angiogenesis factors (LPL, DLL4), extracellular matrix remodeling factors (TGFß, SKIL, LOXL1, IL-1ß, ADAM33, CLEC4A), complement components (C3, CR2) and germinal center activation markers (ZDHHC2 and BLK) compared to controls. MCD showed upregulation of IL-6 (IL-6ST, OSMR and LIFR), IL-2, plasma cell differentiation (XBP1), FDC marker (CXCL13, clusterin), fibroblastic reticular cell cytokine (CCL21), angiogenesis factor (VEGF), and mTORC1 pathway genes compared to UCD and controls. ISH studies demonstrated that VEGF was increased in the follicular dendritic cell-predominant atretic follicles and the interfollicular macrophages of MCD compared to UCD and controls. IL-6 expression was higher along interfollicular vasculature-associated cells of MCD. Immune repertoire analysis revealed oligoclonal expansions of T-cell populations in MCD cases (2/6) and UCD cases (1/9) that are consistent with antigen-driven T cell activation. The findings highlight the unique genes, pathways and cell types involved in UCD and MCD. We identify potential novel targets in CD that may be harnessed for therapeutics.

B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates.

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.

Alternative mechanisms of receptor editing in autoreactive B cells.

Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA V(H) regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the V(H). Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and V(H) replacement.

Autoantibodies targeting cytokines and connective tissue disease autoantigens are common in acute non-SARS-CoV-2 infections.

The widespread presence of autoantibodies in acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is increasingly recognized, but the prevalence of autoantibodies in infections with organisms other than SARS-CoV-2 has not yet been reported. We used protein arrays to profile IgG autoantibodies from 317 samples from 268 patients across a spectrum of non-SARS-CoV-2 infections, many of whom were critically ill with pneumonia. Anti-cytokine antibodies (ACA) were identified in > 50% of patients infected with non-SARS-CoV-2 viruses and other pathogens, including patients with pneumonia attributed to bacterial causes. In cell-based functional assays, some ACA blocked binding to surface receptors for type I interferons (Type I IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). Autoantibodies against traditional autoantigens associated with connective tissue diseases (CTDs) were also commonly observed in these cohorts, including newly-detected antibodies that emerged in longitudinal samples from patients infected with influenza. We conclude that autoantibodies, some of which are functionally active, may be much more prevalent than previously appreciated in patients who are symptomatically infected with diverse pathogens.

A prospective study of influenza vaccination and a comparison of immunologic parameters in children and adults with chromosome 22q11.2 deletion syndrome (digeorge syndrome/velocardiofacial syndrome).

Prior to the advent of cardiac bypass, most children with congenital cardiac anomalies and chromosome 22q11.2 deletion syndrome died. With improved technology, there is now a wave of young adults with chromosome 22q11.2 deletion syndrome requiring clinical care. Fifteen young children and 20 adults with chromosome 22q11.2 deletion had flow cytometry, functional T cell analyses, and functional B cell analyses to characterize their immune system. Subjects were vaccinated with the annual inactivated influenza vaccine, and responses were evaluated by hemagglutination inhibition titer assessment. The pattern of T cell subset abnormalities was markedly different between pediatric and adult patients. In spite of the cellular deficits observed in adults, titers produced after influenza vaccine administration were largely intact. We conclude that disruption to T cell production appears to have secondary consequences for T cell differentiation and B cell function although the clinical impact remains to be determined.

Adoptive transfer of autologous T cells improves T-cell repertoire diversity and long-term B-cell function in pediatric patients with neuroblastoma.

PURPOSE: Children with high-risk neuroblastoma have a poor prognosis with chemotherapy alone, and hematopoietic stem cell transplantation offers improved survival. As a dose-escalation strategy, tandem transplants have been used, but are associated with persistent immunocompromise. This study evaluated the provision of an autologous costimulated, activated T-cell product to support immunologic function. EXPERIMENTAL DESIGN: Nineteen subjects with high-risk neuroblastoma were enrolled in a pilot phase and 23 subjects were entered in to the randomized study. Immunologic reconstitution was defined by flow cytometric and functional assays. Next-generation sequencing was conducted to identify changes to the T-cell repertoire. Twenty-two patients were vaccinated to define effects on antibody responses. RESULTS: Subjects who received their autologous costimulated T-cell product on day 2 had significantly superior T-cell counts and T-cell proliferation compared with those who received T cells on day 90. Early administration of autologous T cells suppressed oligoclonality and enhanced repertoire diversity. The subjects who received the day 2 T-cell product also had better responses to the pneumococcal vaccine. CONCLUSIONS: The infusion of activated T cells can improve immunologic function especially when given early after transplant. This study showed the benefit of providing cell therapies during periods of maximum lymphopenia.

Antibody-mediated rejection in heart transplant recipients: potential efficacy of B-cell depletion and antibody removal.

We present four patients with late AMR following cardiac transplantation, which was associated with de novo post-transplant anti-HLA class II antibody production. All patients had negative anti-HLA class I and class II antibodies prior to transplantation (as assessed by sensitive Flow PRA bead assays) and had a negative retrospective T- and B-cell flow cytometric cross-match. Upon presentation with late graft rejection due to AMR, all patients were treated with rituximab and serial plasmapheresis with IVIg plus triple-drug immunosuppression therapy. Despite initial responses to therapy, relapses occurred in all of the patients and necessitated prolonged or multiple hospital admissions and second transplants in two cases. Post-transplant serum antibody monitoring did not prove to be predictive of treatment success or failure. Serum anti-HLA antibodies should be monitored after heart transplantation. We recommend an assessment of anti-HLA antibodies following a decline in immunosuppressant drug levels or in the presence of heart failure symptoms. Anti-HLA antibody detection should be performed using very sensitive techniques such as microparticle-based assays.

Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells.

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.

Regulation of anti-phosphatidylserine antibodies.

The degree of heavy chain (H) editing, the types of Vkappa editors, and the pattern of Jkappa usage are correlated with a range of the affinity of anti-DNA. This range was determined by the number and location of arginine (R) residues in the VH. We, here, changed a key arginine residue in the VH of anti-DNA transgene to glycine, which sharply reduces the affinity for dsDNA. However, complete reversion of this anti-DNA to germline enhances the affinity for phosphatidylserine (PS). The B cells of this low-affinity anti-DNA and anti-PS transgenic mouse are tightly regulated by receptor editing. Thus, anti-PS B cells are another example of a constitutive self-antigen regulated in the bone marrow.

Duffy antigen receptor and genetic susceptibility of African Americans to acute rejection and delayed function.

BACKGROUND: The unique distribution of the alleles for the Duffy antigen receptor complex (DARC) that binds to chemokines may be associated with the rates of acute rejection and delayed allograft function (DGF) among African Americans. METHODS: A prospective, multicenter cohort study enrolled 222 African American recipients of cadaveric renal allografts from eight adult transplant centers. Subjects were typed by allele-specific polymerase chain reaction (ASPCR) for the polymorphism at position 535 that determines the level of transcription. Associations of DARC genotypes were examined in Cox hazards models with episodes of acute rejection and in logistic regression models with the development of DGF. RESULTS: FyB Null homozygosity was observed among 67% of the recipients. Fifteen percent of the study cohort experienced at least one episode of acute rejection, and the incidence of DGF was 42.5%. The number of FyB Null alleles and FyB Null homozygosity had no detectable association with the rate of acute rejection (P > 0.50) or with the development of DGF (P > 0.50). CONCLUSION: The susceptibility of African American recipients to acute rejection and to DGF was not confirmed to be associated with DARC alleles or genotype. Future studies should exclude a potential role of donor-related DARC in transplant outcomes.