Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma.

BACKGROUND: EGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. METHODS: EGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate 'tumor-monovalent' EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence. RESULTS: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost. CONCLUSION: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue.

Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach.

Antibodies that bind peptide-MHC (pMHC) complex in a manner akin to T cell receptor (TCR) have not only helped in understanding the mechanism of TCR-pMHC interactions in the context of T cell biology but also spurred considerable interest in recent years as potential cancer therapeutics. Traditional methods to generate such antibodies using hybridoma and B cell sorting technologies are sometimes inadequate, possibly due to the small contribution of peptide to the overall B cell epitope space on the surface of the pMHC complex (typical peptide MW = 1 kDa versus MHC MW = 45 kDa) and to the multiple efficiency limiting steps inherent in these methods. In this chapter we describe phage display approaches, including a cell panning strategy, for the rapid generation of such antibodies with high specificity and affinity.

Intraperitoneal Pretargeted Radioimmunotherapy for Colorectal Peritoneal Carcinomatosis.

Peritoneal carcinomatosis (PC) is considered incurable, and more effective therapies are needed. Herein we test the hypothesis that GPA33-directed intracompartmental pretargeted radioimmunotherapy (PRIT) can cure colorectal peritoneal carcinomatosis. Nude mice were implanted intraperitoneally with luciferase-transduced GPA33-expressing SW1222 cells for aggressive peritoneal carcinomatosis (e.g., resected tumor mass 0.369 ± 0.246 g; n = 17 on day 29). For GPA33-PRIT, we administered intraperitoneally a high-affinity anti-GPA33/anti-DOTA bispecific antibody (BsAb), followed by clearing agent (intravenous), and lutetium-177 (Lu-177) or yttrium-86 (Y-86) radiolabeled DOTA-radiohapten (intraperitoneal) for beta/gamma-emitter therapy and PET imaging, respectively. The DOTA-radiohaptens were prepared from S-2-(4-aminobenzyl)-1,4,7, 10-tetraazacyclododecane tetraacetic acid chelate (DOTA-Bn). Efficacy and toxicity of single- versus three-cycle therapy were evaluated in mice 26-27 days post-tumor implantation. Single-cycle treatment ([177Lu]LuDOTA-Bn 111 MBq; tumor dose: 4,992 cGy) significantly prolonged median survival (MS) approximately 2-fold to 84.5 days in comparison with controls (P = 0.007). With three-cycle therapy (once weekly, total 333 MBq; tumor dose: 14,975 cGy), 6/8 (75%) survived long-term (MS > 183 days). Furthermore, for these treated long-term survivors, 1 mouse was completely disease free (microscopic "cure") at necropsy; the others showed stabilized disease, which was detectable during PET-CT using [86Y]DOTA-Bn. Treatment controls had MS ranging from 42-52.5 days (P < 0.001) and 19/20 mice succumbed to progressive intraperitoneal disease by 69 days. Multi-cycle GPA33 DOTA-PRIT significantly prolongs survival with reversible myelosuppression and no chronic marrow (929 cGy to blood) or kidney (982 cGy) radiotoxicity, with therapeutic indices of 12 for blood and 12 for kidneys. MTD was not reached.

Potent ex vivo armed T cells using recombinant bispecific antibodies for adoptive immunotherapy with reduced cytokine release.

BACKGROUND: T cell-based immunotherapies using chimeric antigen receptors (CAR) or bispecific antibodies (BsAb) have produced impressive responses in hematological malignancies. However, major hurdles remained, including cytokine release syndrome, neurotoxicity, on-target off-tumor effects, reliance on autologous T cells, and failure in most solid tumors. BsAb armed T cells offer a safe alternative. METHODS: We generated ex vivo armed T cells (EATs) using IgG-[L]-scFv-platformed BsAb, where the anti-CD3 (huOKT3) scFv was attached to the light chain of a tumor-binding IgG. BsAb density on EAT, in vitro cytotoxicity, cytokine release, in vivo trafficking into tumors, and their antitumor activities were evaluated in multiple cancer cell lines and patient-derived xenograft mouse models. The efficacy of EATs after cryopreservation was studied, and gamma delta (γδ) T cells were investigated as unrelated alternative effector T cells. RESULTS: The antitumor potency of BsAb armed T cells was substantially improved using the IgG-[L]-scFv BsAb platform. When compared with separate BsAb and T cell injection, EATs released less TNF-α, and infiltrated tumors faster, while achieving robust antitumor responses. The in vivo potency of EAT therapy depended on BsAb dose for arming, EAT cell number per injection, total number of EAT doses, and treatment schedule intensity. The antitumor efficacy of EATs was preserved following cryopreservation, and EATs using γδ T cells were safe and as effective as αß T cell-EATs. CONCLUSIONS: EATs exerted potent antitumor activities against a broad spectrum of human cancer targets with remarkable safety. The antitumor potency of EATs depended on BsAb dose, cell number and total dose, and schedule. EATs were equally effective after cryopreservation, and the feasibility of third-party γδ-EATs offered an alternative for autologous T cell sources.

Engineered Cells as a Test Platform for Radiohaptens in Pretargeted Imaging and Radioimmunotherapy Applications.

Pretargeted imaging and radioimmunotherapy approaches are designed to have superior targeting properties over directly targeted antibodies but impose more complex pharmacology, which hinders efforts to optimize the ligands prior to human applications. Human embryonic kidney 293T cells expressing the humanized single-chain variable fragment (scFv) C825 (huC825) with high-affinity for DOTA-haptens (293T-huC825) in a transmembrane-anchored format eliminated the requirement to use other pretargeting reagents and provided a simplified, accelerated assay of radiohapten capture while offering normalized cell surface expression of the molecular target of interest. Using binding assays, ex vivo biodistribution, and in vivo imaging, we demonstrated that radiohaptens based on benzyl-DOTA and a second generation "Proteus" DOTA-platform effectively and specifically engaged membrane-bound huC825, achieving favorable tumor-to-normal tissue uptake ratios in mice. Furthermore, [86Y]Y-DOTA-Bn predicted absorbed dose to critical organs with reasonable accuracy for both [177Lu]Lu-DOTA-Bn and [225Ac]Ac-Pr, which highlights the benefit of a dosimetry-based treatment approach.

A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy.

PURPOSE: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. EXPERIMENTAL DESIGN: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. RESULTS: SADA-BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA-BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. CONCLUSIONS: The SADA-BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system "proteus-DOTA" or "Pr." Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity. Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies.

T cell-bispecific antibodies (BsAbs) couple cytotoxic T lymphocytes to tumor cells, inducing their destruction. Although there are more than 60 classes of BsAbs in development, the relative importance of parameters such as interdomain spacing or spatial configuration is largely unknown. Here, we dissected a symmetric dual bivalent BsAb platform (IgG-[L]-scFv: antitumor IgG with anti-CD3 scFv fused to the light chains) to explore the importance of valency and spatial configuration for BsAb-induced T cell cytotoxicity. Our results revealed that placing tumor and T cell binding domains on the same side of a BsAb (cis-configuration) elicited substantially stronger antitumor activity, in vitro and in vivo, compared to positioning them on opposite sides (trans-configuration). Moreover, using two cis-modules in the same BsAb further improved cytotoxicity (up to 2000-fold). In addition, separating antigen-binding components with a single Ig domain (CL) markedly enhanced cytokine release and in vivo tumor responses compared to smaller (G4S1) or larger (CH1-CH2-CH3) spacers. These findings provide guidelines for improving BsAb function and highlight the importance of spatial configuration and dual bivalency as development parameters.

TCR-mimic bispecific antibodies targeting LMP2A show potent activity against EBV malignancies.

EBV infection is associated with a number of malignancies of clinical unmet need, including Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, and posttransplant lymphoproliferative disease (PTLD), all of which express the EBV protein latent membrane protein 2A (LMP2A), an antigen that is difficult to target by conventional antibody approaches. To overcome this, we utilized phage display technology and a structure-guided selection strategy to generate human T cell receptor-like (TCR-like) monoclonal antibodies with exquisite specificity for the LMP2A-derived nonamer peptide, C426LGGLLTMV434 (CLG), as presented on HLA-A*02:01. Our lead construct, clone 38, closely mimics the native binding mode of a TCR, recognizing residues at position P3-P8 of the CLG peptide. To enhance antitumor potency, we constructed dimeric T cell engaging bispecific antibodies (DiBsAb) of clone 38 and an affinity-matured version clone 38-2. Both DiBsAb showed potent antitumor properties in vitro and in immunodeficient mice implanted with EBV transformed B lymphoblastoid cell lines and human T cell effectors. Clone 38 DiBsAb showed a stronger safety profile compared with its affinity-matured variant, with no activity against EBV- tumor cell lines and a panel of normal tissues, and was less cross-reactive against HLA-A*02:01 cells pulsed with a panel of CLG-like peptides predicted from a proteomic analysis. Clone 38 was also shown to recognize the CLG peptide on other HLA-A*02 suballeles, including HLA-A*02:02, HLA-A*02:04, and HLA-A*02:06, allowing for its potential use in additional populations. Clone 38 DiBsAb is a lead candidate to treat EBV malignancies with one of the strongest safety profiles documented for TCR-like mAbs.

Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach.

Antibodies that bind peptide-MHC (pMHC) complex in a manner akin to T-cell receptor (TCR) have not only helped in understanding the mechanism of TCR-pMHC interactions in the context of T-cell biology, but also spurred considerable interest in recent years as potential cancer therapeutics. Traditional methods to generate such antibodies using hybridoma and B-cell sorting technologies are sometimes inadequate, possibly due to the small contribution of peptide to the overall B-cell epitope space on the surface of the pMHC complex (typical peptide MW = 1 kDa versus MHC MW = 45 kDa), and to the multiple efficiency limiting steps inherent in these methods. In this chapter, we describe a phage display approach for the rapid generation of such antibodies with high specificity and affinity.

Impaired B cell immunity in acute myeloid leukemia patients after chemotherapy.

BACKGROUND: Changes in adaptive immune cells after chemotherapy in adult acute myeloid leukemia (AML) may have implications for the success of immunotherapy. This study was designed to determine the functional capacity of the immune system in adult patients with AML who have completed chemotherapy and are potential candidates for immunotherapy. METHODS: We used the response to seasonal influenza vaccination as a surrogate for the robustness of the immune system in 10 AML patients in a complete remission post-chemotherapy and performed genetic, phenotypic, and functional characterization of adaptive immune cell subsets. RESULTS: Only 2 patients generated protective titers in response to vaccination, and a majority of patients had abnormal frequencies of transitional and memory B-cells. B-cell receptor sequencing showed a B-cell repertoire with little evidence of somatic hypermutation in most patients. Conversely, frequencies of T-cell populations were similar to those seen in healthy controls, and cytotoxic T-cells demonstrated antigen-specific activity after vaccination. Effector T-cells had increased PD-1 expression in AML patients least removed from chemotherapy. CONCLUSION: Our results suggest that while some aspects of cellular immunity recover quickly, humoral immunity is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. The observed B-cell abnormalities may explain the poor response to vaccination often seen in AML patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immunity and increased PD-1 expression shortly after chemotherapy might have implications for the success of several modalities of immunotherapy.

Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors.

Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Generation of TCR-Like Antibodies Using Phage Display.

The adaptive immune response against cancer consists of two arms: the humoral response from B cells, and the cell-mediated response from T cells. The humoral response has the advantage of diversity, theoretically recognizing antigens of any type (sugar, protein, lipid, etc.), but is generally limited to surface-expressed targets. T cells on the other hand, can recognize intracellular targets, but only if they are proteins, and presented as small peptide fragments on major histocompatibility complex (MHC) cell surface antigens. However, with advances in protein engineering and phage display, it has become feasible to quickly identify and generate antibodies or single-chain variable fragments against peptide-MHC, thus bridging the two arms, and allowing for recognition, identification, and effector responses against cells expressing intracellular targets.

Bone marrow plasma cells are a primary source of serum HIV-1-specific antibodies in chronically infected individuals.

Several potent and broadly neutralizing Abs to HIV-1 have been isolated recently from peripheral blood B cells of infected individuals, based on prescreening of Ab activity in the serum. However, little is known regarding the cells that make the Abs that circulate in the blood. Accordingly, we investigated the most likely source, the bone marrow, of chronically HIV-1-infected individuals who were not receiving antiretroviral therapy. Increased frequencies of plasma cells, as well as B cell precursors, namely preB-I and preB-II, and decreased frequencies of mature B cells were observed in bone marrow aspirates of these individuals compared with HIV-negative counterparts. Increased frequencies of bone marrow plasma cells are consistent with known hallmarks of HIV-1 infection, namely hypergammaglobulinemia and increased frequencies of peripheral blood plasmablasts. Levels of HIV-1 envelope (Env)-binding and HIV-1-neutralizing Abs were measured in serum, and corresponding frequencies of Ab-secreting or Env-binding cells were measured in the blood (plasmablasts and memory B cells) and in the bone marrow (plasma cells). A strong correlation was observed between serum HIV-1-specific Abs and Env-specific bone marrow-derived plasma cells, but not circulating plasmablasts or memory B cells. These findings demonstrate that, despite HIV-1-induced phenotypic and functional B cell dysregulation in the peripheral blood and secondary lymphoid tissues, bone marrow plasma cells remain a primary source for circulating HIV-1-specific Abs in HIV-1-infected individuals.

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals.

Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.

Graves' disease as immune reconstitution disease in HIV-positive patients is associated with naive and primary thymic emigrant CD4(+) T-cell recovery.

OBJECTIVE: Immune restoration disease (IRD) can develop in HIV-infected patients following antiretroviral therapy (ART) initiation as unmasking or paradoxical worsening of opportunistic infections and, rarely, autoimmune phenomena. Although IRD usually occurs in the first months of ART during memory CD4 T-cell recovery, Graves' disease occurs as a distinctive late-onset IRD and its pathogenesis is unclear. DESIGN: Seven patients who developed Graves' disease following ART initiation from the primary HIV care clinic at the National Institutes of Health were retrospectively identified and each was matched with two HIV-infected controls based on age, sex, and baseline CD4 T-cell count. Laboratory evaluations on stored cryopreserved samples were performed. METHODS: Immunophenotyping of peripheral blood mononuclear cells (PBMCs), T-cell receptor excision circle (TREC) analysis in PBMCs, measurement of serum cytokines, and luciferase immunoprecipitation systems (LIPS) analysis for autoimmune antibodies were performed on stored samples for cases and controls at baseline and longitudinally following ART initiation. TSH/thyrotropin receptor (TSH-R) antibody testing was performed on serum from cases. Data were analyzed using nonparametric testing. RESULTS: In comparison with controls, the proportion of naive CD4 T cells increased significantly (P = 0.0027) in the Graves' disease-IRD patients. TREC/10 PBMCs also increased significantly following ART in Graves' disease-IRD patients compared with controls (P = 0.0071). Similarly, LIPS analysis demonstrated increases in nonthyroid-related autoantibody titers over time following ART in cases compared with controls. CONCLUSION: Our data suggest that Graves' disease-IRD, in contrast to early-onset IRD, is associated with naive and primary thymic emigrant CD4 T-cell recovery and inappropriate autoantibody production.

CXCR4/IgG-expressing plasma cells are associated with human gastrointestinal tissue inflammation.

BACKGROUND: We previously reported abnormalities in circulating B cells in patients with chronic granulomatous disease (CGD) and those with HIV infection. Gastrointestinal complications are common to both diseases and likely involve perturbation of immune cells, including plasma cells (PCs). IgA is the most abundant immunoglobulin in the human body, with roles in protection and maintenance of intestinal homeostasis. IgA is produced primarily by PCs residing in mucosal tissues that are also thought to circulate in the blood. OBJECTIVE: We sought to characterize and compare PCs in patients with infectious (HIV) and noninfectious (CGD and Crohn disease) diseases that have been associated with intestinal inflammation. METHODS: Phenotypic and transcriptional analyses were performed on cells isolated from the blood and colon. RESULTS: IgA-secreting CCR10-expressing PCs predominated in the guts of healthy subjects, whereas in patients with HIV, CGD, and Crohn disease, there was a significant increase in the proportion of IgG-secreting PCs. Where intestinal inflammation was present, IgG-secreting PCs expressed reduced levels of CCR10 and increased levels of CXCR4. The intensity of CXCR4 expression correlated with the frequency of IgG-expressing PCs and the frequency of CXCR4(+)/IgG(+) PCs was associated with the severity of intestinal inflammatory disease yet distinct from PCs and plasmablasts circulating in the blood. CONCLUSIONS: These findings suggest that regardless of the underlying disease, the presence of CXCR4(+)/IgG(+) PCs in the gut is a strong yet localized indicator of intestinal inflammation. Furthermore, our findings suggest that CXCR4(+)/IgG(+) PCs might play a role in immune cell homeostasis during inflammatory processes of the gut.

Humans with chronic granulomatous disease maintain humoral immunologic memory despite low frequencies of circulating memory B cells.

CD27(+) memory B cells are reduced in the blood of patients with chronic granulomatous disease (CGD) for reasons and consequences that remain unclear. Here we confirm not only decreased CD27(+) but also IgG(+) B cells in the blood of CGD patients compared with healthy donors (HDs). However, among IgG(+) B cells, the ratio of CD27(-) to CD27(+) was significantly higher in CGD patients compared with HDs. Similar to conventional memory B cells, CD27(-)IgG(+) B cells of CGD patients expressed activation markers and had undergone somatic hypermutation, albeit at levels lower than their CD27(+) counterparts. Functional analyses revealed slight reductions in frequencies of total IgG but not influenza-specific memory B-cell responses, as measured by Elispot in CGD patients compared with HDs. Serum IgG levels and influenza-specific antibodies were also normal in these CGD patients. Finally, we provide evidence that influenza-specific memory B cells can be present within the CD27(-)IgG(+) B-cell compartment. Together, these findings show that, despite reduced circulating CD27(+) memory B cells, CGD patients maintain an intact humoral immunologic memory, with potential contribution from CD27(-) B cells.