B-cell Exhaustion Research Articles

Abstract 6869: Paired single-cell B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cell immunity in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the second most common adult leukemia with a five-year survival rate around 30%. For decades, AML treatment has centered around intensive chemotherapy frequently followed by allogeneic stem cell transplant (Allo-SCT). The power of Allo-SCT to induce lasting remission has been attributed to a potent graft-versus-leukemia effect and has inspired efforts to stimulate host immunity against AML via immune checkpoint blockade (ICB). Thus far, these studies have yielded mixed results. While tumor-infiltrating T-cells have long been the focus of immunotherapy investigations, a growing body of data points to a central role of B-cells in mediating anti-tumor immunity, and a comprehensive assessment of B lymphocytes within the AML immune microenvironment has never been carried out with single cell clonal resolution. We performed paired single-cell transcriptomic and B cell receptor (BCR) analysis on 52 bone marrow aspirate samples. These samples included 6 from healthy bone marrow donors (normal), 24 from newly diagnosed AML patients (NewlyDx), and 22 from 8 relapsed or refractory AML patients (RelRef), who underwent assessment both before and after ICB with azacitidine/nivolumab. We first used transcriptomic data to delineate canonical B-cell subpopulations and observed a reduction in nascent B cells and an expansion of differentiated B cells in AML patients (RelRef>NewlyDx). Overlaying BCR sequencing and clonotypic analysis revealed clonal expansion, low clonotypic diversity, and extensive somatic hypermutation in the RelRef samples, suggesting antigen-driven affinity maturation within the tumor microenvironment. Furthermore, we identified robust AP-1 expression (a marker of activation) in B-cells from NewlyDx patients and a marked loss of AP-1 in RelRef patients, suggesting that B-cell exhaustion may be a key immunologic feature of AML relapse. Importantly, we observed that AP-1 activity is restored and that specific B-cell clones expand after ICB, but only among patients who clinically respond to ICB. In line with these observations, we also noted the clonal expansion of AML-associated plasma cells and loss of clonotypic diversity among ICB clinical responders, pointing to an antitumor role for these clones. Finally, we characterized atypical memory B cells which demonstrate high antigen presentation activity, close interaction with AML cells and are associated with unfavorable clinical outcomes, suggesting that they play a direct role in aiding AML survival and immune escape. Collectively, our findings establish the dynamic landscape of AML-associated B-cells while identifying specific B-cell subpopulations and B-cell-AML interactions that may be targeted by the next generation of AML-directed immunotherapies. Citation Format: Shengnan Guo, Gopi S. Mohan, Bofei Wang, Tianhao Li, Naval Daver, Yuting Zhao, Patrick K. Reville, Dapeng Hao, Hussein A. Abbas. Paired single-cell B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cell immunity in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6869.

MYC to BCL6 State-Transitions Determine Cell Size and Metabolic Fluctuations and Define a Novel Biorhythm in B-Cell Malignancies

Background: Cell size fluctuations are well-documented during critical transitions during normal B-cell development. For instance, during early stages of development, “large cycling pre-B cells” (also classified as Fraction C') eventually exit cell cycle and shrink to become “small resting pre-B cells” (Fraction D) to enable immunoglobulin light chain gene recombination. We and others had shown that this transition is marked by loss of Myc expression and gain of Bcl6. Later in development, germinal center B-cells cycle between large MYC+ “centroblast” and smaller BCL6+ “centrocyte” states between dark and light zones, respectively. MYC is involved in biomass accumulation and provides fuel for cell division, while BCL6 confers a quiescent phenotype to cells and protects B cells from DNA damage-induced apoptosis. BCL6 can suppress MYC transcription, indicating MYC and BCL6 is mutually exclusive, and their expression dictates distinct cellular states. Significance: In B-cell malignancies, transitions between mutually exclusive MYC+ and BCL6+ states seem to be important as well: B-ALL cells that are driven by oncogenic tyrosine kinases (e.g. BCR-ABL1) express predominantly MYC but can be forced into a BCL6+ state upon tyrosine kinase inhibition (e.g. imatinib). In germinal center-derived B-cell lymphoma, both MYC and BCL6 are frequently targeted by chromosomal translocation, suggesting that disruption of physiological state transitions may be part of the malignant transformation program. Results: To elucidate regulation of MYC-BCL6 state transitions and their importance in progression and development of drug resistance in B-ALL and germinal center-derived B-cell lymphoma, we developed a MYC-eGFP and BCL6-mCherry dual-reporter mouse model. In addition, we engineered human B-ALL PDX cells with MYC-mNeonGreen and BCL6mScarlet knockin fusion genes by CRISPR/Cas9-mediated HDRT. As expected, MYC+ and BCL6+ states were largely mutually exclusive during early stages of B-cell development, as well as in BCR-ABL1- and NRAS G12D-driven B-ALL. Interestingly, a large fraction of B-ALL cells expressed neither MYC nor BCL6, as validated by cell sorting and Western blot. Based on single-cell sorting and subsequent time-lapse monitoring over 12 hours, we found MYC+ cells transitioning through a double-negative state to become BCL6+ and then revert again to a MYC+ state ( Figure A), suggesting dynamic transitions between MYC and BCL6 cycles. We characterized these populations by analyzing cell size, cell proliferation, cell cycle, clone formation, and gene expression profiles. The results indicated that MYC+ cells exhibited larger cell size, active proliferation, and increased glycolytic activity. Conversely, BCL6+ cells displayed smaller cell size, activation of autophagy with suppression of glycolysis, and cell cycle arrest in the G0 phase of the cell cycle. Conclusions and future directions: Given the dynamic inter-transition between MYC and BCL6 states, and the significant roles of both MYC and BCL6 in B-ALL and germinal center-derived B-cell lymphomas, we propose MYC- and BCL6-dependent fluctuations of cellular activity (wake) and quiescence (sleep; Figure B). Thereby, MYC and BCL6 are expressed in an oscillatory manner. During the MYC state, cells exhibit high glycolysis activity, actively accumulate biomass, and undergo proliferation, representing the wake phase. In contrast, during the BCL6 state, cells suppress glycolysis metabolism, pause the cell cycle, and display a quiescent phenotype, representing the sleep phase. Based on this discovery, we will examine the ‘B-cell exhaustion’ paradigm that Bcl6 and Myc mark iterative cycles of quiescence and activation. In analogy to sleep-wake phases, we hypothesize that the Bcl6-autophagy phase is essential for recovery and regeneration. We will investigate how B-cells transition from one phase to the next and consequences of elongation of one phase at the expense of the other. Malignant B-cells may be particularly sensitive to shortening of quiescence periods (“sleep deprivation”). These and other observations will lead to new conceptual frameworks for the understanding of how MYC-BCL6 state transitions, cell-size fluctuations and the length of recovery-periods regulate energy-supply and survival of B-cell malignancies and create opportunities for therapeutic intervention.

NK-cell exhaustion, B-cell exhaustion and T-cell exhaustion-the differences and similarities.

T-cell exhaustion has been extensively researched, compared with B-cell exhaustion and NK-cell exhaustion, which have received considerably less attention; and there is less of a consensus on the precise definitions of NK-cell and B-cell exhaustion. NK-cell exhaustion, B-cell exhaustion and T-cell exhaustion are examples of lymphocyte exhaustion, and they have several differences and similarities. Lymphocyte exhaustion is also frequently confused with anergy, cellular senescence and suppression, because these conditions can have significant overlapping similarities with exhaustion. An additional source of confusion is due to the fact that lymphocyte exhaustion is not a binary state, but instead has a spectrum of severity induced by different levels and duration of continuous antigenic stimulation. Concurrent multiple types of lymphocyte exhaustion are possible, and this situation is herein called poly-lymphocyte exhaustion. Poly-lymphocyte exhaustion for the same cancer or pathogen would be especially dangerous. As there are significant advantages for a pathogen by inducing poly-lymphocyte exhaustion in an immune system, there are pathogens with an evolved capability to induce poly-lymphocyte exhaustion. These pathogens may include certain manipulative viruses, bacteria, fungi and protozoan parasites.

Potency of HIV-2-specific antibodies increase in direct association with loss of memory B cells.

: Potent HIV-neutralizing antibodies are critical for vaccination and viral reservoir control. High levels of neutralizing antibodies characterize HIV-2 infection, a naturally occurring model of attenuated HIV disease with low-to-undectable viremia. We found that HIV-2-specific antibody potency increased in direct association with the loss of both switched and unswitched memory B cells in untreated HIV-2 infection. Thus, HIV antibody affinity maturation is linked to memory B-cell exhaustion even in reduced viremia settings.

Chronic HIV-1 Infection Induces B-Cell Dysfunction That Is Incompletely Resolved by Long-Term Antiretroviral Therapy.

To determine the effect of long-term antiretroviral therapy (ART) on HIV-1-induced B-cell dysfunction. Comparative study of ART-naive and ART-treated HIV-infected patients with non-HIV controls. B-cell dysfunction was examined in patients with HIV-1 infection (n = 30) who had received ART for a median time of 9.25 years (range: 1.3-21.7) by assessing proportions of CD21 B cells (a marker of B-cell exhaustion) and proportions of tumor necrosis factor-related apoptosis-inducing ligand or B and T lymphocyte attenuator B cells, and serum levels of immunoglobulin free light chains (markers of B-cell hyperactivation). The association of these markers with serum levels of IgG1 and IgG2, and production of IgG antibodies after vaccination with pneumococcal polysaccharides were also examined. ART-naive patients with HIV (n = 20) and controls (n = 20) were also assessed for comparison. ART-treated patients had increased proportions of CD21 and tumor necrosis factor-related apoptosis-inducing ligand B cells and, furthermore, although proportions of B and T lymphocyte attenuator B cells were not significantly different from controls, they correlated negatively with CD21 B cells. Proportions of CD21 B cells also correlated negatively with current CD4 T-cell counts. In ART-naive patients with HIV, free light chains correlated with CD21 B cells and IgG1, but not IgG2. Serum IgG2:IgG1 ratios were substantially lower than normal in patients with HIV and did not resolve on ART. In ART-treated patients, IgG antibody responses to pneumococcal polysaccharides after vaccination were not associated with markers of B-cell dysfunction. B-cell dysfunction persists in patients with HIV receiving long-term ART. The causes and consequences of this require further investigation.

Association between malaria exposure and Kaposi's sarcoma-associated herpes virus seropositivity in Uganda.

ObjectiveUnlike other herpes viruses, Kaposi's sarcoma-associated herpes virus (KSHV) is not ubiquitous worldwide and is most prevalent in sub-Saharan Africa. The reasons for this are unclear. As part of a wider investigation of factors that facilitate transmission in Uganda, a high prevalence country, we examined the association between antimalaria antibodies and seropositivity against KSHV.MethodsAntibodies against P. falciparum merozoite surface protein (PfMSP)-1, P. falciparum apical membrane antigen (PfAMA)-1 and KSHV antigens (ORF73 and K8.1) were measured in samples from 1164 mothers and 1227 children.ResultsKaposi's sarcoma-associated herpes virus seroprevalence was 69% among mothers and 15% children. Among mothers, KSHV seroprevalence increased with malaria antibody titres: from 60% to 82% and from 54% to 77%, comparing those with the lowest and highest titres for PfMSP-1 and PfAMA-1, respectively (P < 0.0001). Among children, only antibodies to PfAMA-1 were significantly associated with KSHV seropositivity, (P < 0.0001). In both mothers and children, anti-ORF73 antibodies were more strongly associated with malaria antibodies than anti-K8.1 antibodies.ConclusionThe association between malaria exposure and KSHV seropositivity suggests that malaria is a cofactor for KSHV infection or reactivation.

B-cell exhaustion in HIV infection: the role of immune activation.

To discuss a component of the pathogenic mechanisms of HIV infection in the context of phenotypic and functional alterations in B cells that are due to persistent viral replication leading to aberrant immune activation and cellular exhaustion. We explore how B-cell exhaustion arises during persistent viremia and how it compares with T-cell exhaustion and similar B-cell alterations in other diseases. HIV-associated B-cell exhaustion was first described in 2008, soon after the demonstration of persistent virus-induced T-cell exhaustion, as well as the identification of a subset B cells in tonsil tissues with immunoregulatory features similar to those observed in T-cell exhaustion. Our understanding of B-cell exhaustion has since expanded in two important areas: the role of inhibitory receptors in the unresponsiveness of exhausted B cells and the increasing evidence that similar B cells are found in other diseases that are associated with aberrant immune activation and inflammation. The phenomenon of B-cell exhaustion is now well established in HIV infection and other diseases characterized by immune activation. Over the coming years, it will be important to understand how cellular exhaustion affects the capacity of the immune system to respond to persisting primary pathogens, as well as to other microbial antigens, whether encountered as secondary infections or following immunization.

Peripheral CD27-CD21- B-cells represent an exhausted lymphocyte population in hepatitis C cirrhosis.

Hepatitis C cirrhosis is associated with a profound disappearance of memory B-cells. We sought to determine if this loss is associated with the expansion of the CD27(-)CD21(-) tissue-like memory B-cells with features of B-cell exhaustion. To this end, we quantified the frequency of CD27(-)CD21(-) B-cells in healthy, non-cirrhotic HCV-infected, and cirrhotic patients. We examined the expression of putative inhibitory receptors, the proliferative and immunoglobulin-secreting capacity of CD27/CD21-defined B-cell subsets upon B-cell receptor and/or CD40 stimulation. We found that CD27(-)CD21(-) B-cells are significantly increased in frequency relative to healthy donors in HCV-infected patients. CD27(-)CD21(-) B-cells were hypoproliferative relative to naïve and resting memory B-cells upon agonistic stimulation, but retained similar capacity for antibody secretion. CD27(-)CD21(-) tissue-like memory B-cells with exhausted proliferation circulate at increased frequency in cirrhotic and non-cirrhotic HCV-infected patients. This B-cell subset does not appear anergic, exhibiting immunoglobulin-secreting capacity on CD40 agonism indistinguishable from other CD27/CD21-defined B-cell subsets.

Peripheral B-cell activation and exhaustion markers in patients with ankylosing spondylitis

AimsAnkylosing spondylitis (AS) is an autoimmune disease and is associated with abnormal B cell function. However, the roles of different B cell subsets are less clear. This study aimed to examine the frequency of different subsets of B cells in AS patients following standard therapies. Main methodsEighteen newly diagnosed AS patients and 10 healthy controls (HC) were recruited in this study. The expression of CD27, CD38, CD86, CD95 and IgD on CD19+ B cells was examined by flow cytometry. The disease activity was scored according to the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Serum levels of C-reactive protein (CRP), rheumatoid factor (RF), IgG, IgA and IgM, and the erythrocyte sedimentation rate (ESR) were measured. Key findingsThe frequency of CD27+ B cells was decreased in AS patients compared with HC (p=0.018), while CD86+ and CD27−CD95+ B cell subsets increased in AS patients (p<0.001). Meanwhile, the frequencies of CD38+ and CD95+ B cell positively correlated with BASDAI (r=0.6505, p=0.0035; r=0.6854, p=0.0017, respectively), while CD38−CD86+ B cell negatively correlated with BASDAI (r=−0.7329, p<0.001). We also found that CD27− and CD95+ B cell negatively correlated with RF levels (r=−0.5141, p=0.0290; r=−0.4944, p=0.0370, respectively), while CD27+ B cell positively correlated with IgG levels (p=0.0148, r=0.5640). Moreover, CD86+ and CD27−CD95+ B cell subsets increased following treatment with Meloxicam and Etanercept for one month (p<0.001; p<0.001). SignificanceThese findings suggest that CD27−CD95+CD19+ and CD86+CD19+ B cells may be reasonable cellular targets for the therapeutic intervention of AS.

B cell exhaustion in sickle cell disease patients is associated with inhibited memory generation after influenza vaccination (P3009)

Abstract Dysregulated immunity is a hallmark of Sickle Cell Disease (SCD), and clinical evidence indicates that SCD patients mount adequate initial antigen-specific antibody responses only to have significant drops in titer levels 1-2 years post-vaccination. We thus hypothesized that SCD patients do not generate sufficient B-cell memory after vaccination. We conducted a translational study (IRB #11-026-1) using flow cytometric analysis of memory B-cells from frozen PBMCs taken from 11 SCD patients and 9 healthy controls before and 4-6 weeks after receiving the influenza vaccine. Percentages of CD19+ B-cells were similar between groups before vaccination, but only SCD patients showed a significant increase in B-cells after vaccination (P=0.04). However, control subjects generated approximately 2-fold more CD27+ memory B-cells after vaccination (P=0.03) while almost no change was observed in the SCD patients. This indicates that SCD patients generate naïve, but little to no memory B-cells after vaccination, thereby explaining the diminishing antibody titers observed by clinicians. Further analysis demonstrated that SCD patients have a significantly higher proportion of FcRL4+ exhausted B-cells at baseline when compared to controls (33% more; P=0.02). Given that FcRL4 down-regulates BCR signaling and is associated with reduced memory B-cell generation in people with chronic infections, we propose that chronic inflammation in SCD leads to B-cell exhaustion through BCR signal dampening.

Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection

To define the impact of chronic viremia and associated immune activation on B-cell exhaustion in HIV infection. Progressive HIV infection is marked by B-cell anergy and exhaustion coupled with dramatic hypergammaglobulinemia. Although both upregulation of CD95 and loss of CD21 have been used as markers of infection-associated B-cell dysfunction, little is known regarding the specific profiles of dysfunctional B cells and whether persistent viral replication and its associated immune activation play a central role in driving B-cell dysfunction. Multiparameter flow cytometry was used to define the profile of dysfunctional B cells. The changes in the expression of CD21 and CD95 were tracked on B-cell subpopulations in patients with differential control of viral replication. : Although the emergence of exhausted, CD21 tissue-like memory B cells followed similar patterns in both progressors and controllers, the frequency of CD21 activated memory B cells was lower in spontaneous controllers. Our results suggest that the loss of CD21 and the upregulation of CD95 occur as separate events during the development of B-cell dysfunction. The loss of CD21 is a marker of B-cell exhaustion induced in the absence of appreciable viral replication, whereas the upregulation of CD95 is tightly linked to persistent viral replication and its associated immune activation. Thus, these dysfunctional profiles potentially represent two functionally distinct states within the B-cell compartment.

Rb Protects B-Lineage Hematopoietic Progenitor Cells From Oxidative Stress and Exhaustion

Abstract 1315 Background:Multiple myeloma (MM) is the second most common hematologic malignancy in the United States. Chromosome 13 deletions are found in approximately one half (50%) of all patient samples and is associated with worse outcomes. The retinoblastoma gene (Rb1) has been implicated as a candidate tumor suppressor at 13q14, however, unmutated Rb1 remains expressed from retained alleles in the majority of cases. We sought to explore the role of Rb1 in plasma cells by generating mice with deficiency of Rb1 targeted to late B-cell development. Methods:The Rb family member p107 can compensate for Rb1 function, so we crossed Rb1Flox/Flox and P107−/−mouse strains with Cγ1-Cre knock-in mice that express the Cre recombinase specifically in germinal center (GC) B-cells to generate mice with tissue-specific deletion of Rb in germinal center cells (Cg1-Rb-KO), and we aged these mice to assess disease development. Results:In Cg1-Rb-KO mice, we demonstrated the Rb1 locus was successfully recombined in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). Recombination was also detectable in bone marrow B-cell but not myeloid cell progenitors. Cg1-Rb-KO mice developed normally but became ill starting at 40 weeks of age. Sick mice demonstrated weight loss, hypoplastic spleens, osteopenia and significant lymphopenia. Thorough analysis of bone marrow and spleen hematopoietic progenitor cell subsets by multi-parameter flow cytometry at the time of death revealed dramatic reductions in marrow B-cell (Hardy fractions C-D, from 3.18×103to 40 cells and from 1.4×106 to 7.5×104cells respectively), suggesting an effective collapse of the B-cell lineage in mice lacking Rb. Examination of Cg1-Rb-KO mice prior to becoming sick (age 30–35 weeks) demonstrated increased numbers of B-cell progenitors in the bone marrow, consistent with Rb’s known role in cell cycle regulation. Together, our results suggest Cg1-Rb-KO mice develop benign B-cell hyperplasia followed by a lethal collapse of the B-cell lineage. To help explain this B-cell exhaustion phenotype, we measured reactive oxygen species (ROS) in the B-cell of young Cg1-Rb-KO mice. We observed significantly increased ROS in Cg1-Rb-KO mice at baseline and following LPS stimulation ex vivo and in vivo compared to p107-KO control mice, suggesting that B-cells from Cg1-Rb-KO mice have elevated levels of oxidative stress. Conclusion:Rb1 is important for maintenance of B-cell homeostasis and protects B-lineage progenitor cells from oxidative stress and exhaustion. Rb1 deletion was not sufficient for B-cell transformation and additional mutations are likely required for MM development in the context of complete RB1 loss. Disclosures:Vij:Millennium: Speakers Bureau.

FcRL4 acts as an adaptive to innate molecular switch dampening BCR signaling and enhancing TLR signaling

AbstractFc receptor–like 4 (FcRL4) is expressed on the surface of a subset of memory B cells (MBCs) located at sites of invading pathogens in mucosal lymphoid tissues in healthy individuals. Recently, FcRL4+ MBCs were shown to be greatly increased in number in the peripheral blood of HIV-infected viremic individuals, in whom they are associated with B-cell exhaustion, and in individuals chronically reinfected with malaria. In the present study, we provide evidence that the expression of FcRL4 in human B-cell lines disrupts immune synapse formation and blocks antigen-induced BCR signaling at the point of Syk phosphorylation, blocking downstream activation of PLC-γ2 and Vav and the induction of calcium responses and CD69 expression. FcRL4 functions by ligation-independent mechanisms that require the 3 tyrosine residues in its cytoplasmic domain and involves its phosphorylation and association with the tyrosine phosphatases SHP-1 and SHP-2. Remarkably, FcRL4 is concentrated in endosomes after treatment with the TLR9 agonist CpG and enhances signaling through TLR9, as measured by increased expression of CD23. These findings suggest that FcRL4 may act as a molecular switch in B cells to dampen adaptive immune signaling and enhance innate signaling in response to chronic antigenic stimulation.

Alterations of the B-cell response by HIV-1 replication.

While the hallmark of HIV-1 infection is the progressive depletion of CD4(+) T cells, extensive B-cell dysfunction ensues that impairs the quality of the humoral response. HIV-1 infection causes hypergammaglobulinemia, polyclonal activation, loss of memory B-cell subsets, B-cell exhaustion, aberrant B-cell surface markers, and impaired humoral responses against infections and vaccinations. The totality of the mechanisms that contribute to B-cell dysfunction in vivo is unknown, although roles for HIV proteins (Env, Tat, and Nef) and virions binding to CD21 on B cells have been identified. Recent studies suggest that early antiretroviral therapy, that minimizes virus replication, can profoundly preserve the early B-cell response to HIV-1. Thus, it is clear that there is an intricate interplay between HIV replication and stimulation of the host B-cell response to infection. A better understanding of how HIV-1 subverts a productive B-cell response is needed to inform vaccine strategies that aim to elicit long-lived plasma cells and memory B-cell responses that can act quickly upon antigen stimulation.

Beta-cell mass plasticity in type 2 diabetes.

Prospective studies have shown that insulin resistance (IR) and impaired insulin secretion are independent predecessors and predictors of overt diabetes in genetically predisposed individuals [1–3]. Studies involving subjects at high risk of progression to type 2 diabetes (T2D) have shown that b-cell function begins to decline prior to presentation of elevated, fasting plasma glucose levels, thus predicting the subsequent development of T2D [4]. With the onset of IR, rates of both insulin biosynthesis and secretion are amplified, coupled with increases in b-cell mass and hypertrophy of existing b-cells to meet the increase in demand [5]. As the b-cell mass reaches its threshold to compensate, the burden of IR can no longer be overcome [6]. In those individuals with a genetic predisposition, this lack of pancreatic plasticity ultimately leads to hyperglycaemia [7]. After years of controversy regarding the primacy of either a b-cell defect or IR in the pathogenesis of T2D, we now recognize that various degrees of both alterations must coexist for hyperglycaemia to become manifest. While much is known about the mechanisms of IR, the causes for b-cell defects remain poorly understood. Still more enigmatic is the clinical relationship of the functional b-cell defect to actual b-cell mass. Vague terms such as b-cell exhaustion and fatigue, b-cell rest and survival and b-cell preservation provide little direction towards the effective therapeutic strategies for preserving b-cell function and mass. As the incidence of T2D continues to increase at an alarming rate, it is becoming imperative that the clinical focus shift towards the preservation of b-cell function and mass. Widespread efforts are being undertaken in an attempt to understand the mechanisms that regulate b-cell mass and to determine precisely what goes awry in T2D. Clinicians, however, are limited by the fact that there is no method for monitoring b-cell mass. In the following review, we discuss factors that influence b-cell mass and potential strategies for therapeutic intervention in the preservation of b-cell mass.

Immunopathologic effects associated with Sarcocystis neurona-infected interferon-gamma knockout mice.

Interferon-gamma knockout (IFN-gamma KO) mice were infected with Sarcocystis neurona merozoites to characterize the immunopathology associated with infection. By day 14 postinfection (PI), mice developed splenomegaly and lymphadenopathy, characterized by marked lymphoid hyperplasia with increased numbers of germinal centers. Additional histopathologic changes included increased extramedullary hematopoiesis, multifocal mixed inflammatory infiltrates in the liver, perivascular infiltrate of the liver and lung, and interstitial pneumonia. The total number of B-cell splenocytes (P < 0.05) and the percentage of B-cells increased on day 14 PI in the spleen and on day 28 PI in the lymph nodes (P < 0.05). By day 28 PI, the number of B-cell splenocytes decreased significantly. A non-subset-specific decrease in percentages of CD4 lymphocytes throughout all lymphoid organs was observed on day 14 PI. However, total CD4 and CD44/CD4 splenocytes increased significantly by day 28 PI. Early-activation CD8 lymphocytes were reduced in the blood and spleen, whereas memory CD8 lymphocyte percentages and total numbers were significantly increased. On the basis of the results, we propose that S. neurona-infected IFN-gamma KO mice are immunocompromised and unable to clear the infection. Thus, they develop B-cell exhaustion and a delayed, but sustained, increased number of memory CD4 and CD8 lymphocytes due to chronic antigen stimulation.

Natural history of B-cell dysfunction in spontaneously diabetic Chinese hamsters

To elucidate the pathogenesis of diabetes in spontaneously diabetic Chinese hamsters (CHAD strain), a longitudinal study from just after weaning to overt diabetic state was performed. Fasting and non-fasting plasma glucose, non-fasting plasma insulin and pancreatic hormone contents (insulin, glucagon and amylin) were measured, and light microscopic examination of pancreatic islets by immunohistochemical technique and pancreas perfusion study were performed. No insulitis was found in the islets of the CHAD strain. In animals aged 1 month, there was no significant difference in the percentage of B-cell area to islet area between the CHAD strain and the control. At this stage, hyperinsulinemia was observed despite normal plasma glucose levels both in fasting and non-fasting states. In the animals of the CHAD strain aged 2–4 months, insulin secretion from the pancreas, pancreatic insulin content and non-fasting plasma insulin level decreased in proportion to the decrease of B-cell mass. In animals aged about ten months, severe hyperglycemia and hypoinsulinemia were observed. We demonstrated the existence of amylin-like immunoreactivity in the B-cells of Chinese hamsters. However, no amyloid deposit was observed in the islets of the CHAD strain. After the onset of diabetes, amylin secretion from the pancreas and pancreatic amylin content in the CHAD strain were significantly lower than those in the control. We demonstrated the natural history of B-cell dysfunction in the CHAD strain. It could mean the process of B-cell exhaustion. The profile of the CHAD strain is similar to some types of human NIDDM. Therefore, the CHAD strain is a useful diabetic model in the study of NIDDM.

Marked reduction of pancreatic insulin content in male ventromedial hypothalamic-lesioned spontaneously non-insulin-dependent diabetic (Goto-Kakizaki) rats

The effects of ventromedial hypothalamic (VMH) lesions were examined in male and female non-obese non-insulin-dependent diabetic (Goto-Kakizaki [GK]) rats with respect to glucose metabolism and pancreatic insulin content. VMH lesions produced hyperphagia and hyperinsulinemia in both male and female GK rats. In male rats, plasma glucose levels of VMH-lesioned GK rats (22.7 ± 3.1 mmol/L) were significantly greater than the levels of sham-operated GK rats (10.6 ± 1.0 mmol/L, P < .001) at 7 weeks after the operation, although there were no differences in these levels between VMH-lesioned and sham-operated groups in Wistar rats. Plasma insulin levels in male VMH-lesioned GK rats tended to be lower at 7 weeks than at 1 week. VMH lesions caused a significant decrease in the pancreatic insulin content of male GK rats (12.0 ± 2.3 nmol/L/g pancreas) compared with male sham-operated rats (15.8 ± 1.4 nmol/L/g pancreas, P < .05) 9 weeks postoperatively. In contrast to the results in male rats, female GK rats showed no differences in plasma glucose levels between VMH-lesioned and sham-operated groups at 7 weeks. Female VMH-lesioned GK rats also showed no difference in plasma insulin levels between 1 week and 7 weeks. The pancreatic insulin level of female VMH-lesioned GK rats was unchanged from that of female sham-operated GK rats. The insulin content was significantly greater in the VMH-lesioned Wistar group than in the sham-operated Wistar group, regardless of sex. These observations suggest that the increased requirement for insulin secretion caused by VMH lesions may lead to B-cell exhaustion, producing, at least in part, marked hyperglycemia in male GK rats. Male VMH-lesioned GK rats might be a good model for obese Japanese patients with the genetic predisposition to non-insulin-dependent diabetes mellitus (NIDDM).

Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells.

Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal peptide hormone that is released in response to oral nutrients and that potently augments glucose-mediated insulin secretion. GLP-I(7-37) has potent insulin-releasing activities in vivo in response to oral nutrients, in situ in the isolated perfused pancreas, and in vitro in cultured pancreatic B-cells. As such GLP-I(7-37) is a potent hormonal mediator in the enteroinsular axis involved in the regulation of glucose homeostasis. We now show that in addition to stimulating the release of insulin, GLP-I(7-37) stimulates proinsulin gene expression at the levels of gene transcription and cellular levels of proinsulin messenger RNA as well as the translational biosynthesis of proinsulin. These findings of the positive anabolic actions of GLP-I(7-37) on the synthesis of insulin in B-cells support the notion that GLP-I(7-37) may be of therapeutic use in stimulating the production of insulin in patients with noninsulin-dependent diabetes mellitus and that overproduction of insulin with subsequent hypoglycemia will not occur in response to the administration of GLP-I(7-37). Furthermore, these positive actions of GLP-I(7-37) on insulin production obviate the possibility of B-cell exhaustion in response to such a potent secretagogue.

A mechanism by which primary or secondary hypothalamic involvement results in the development of insulin-dependent diabetes mellitus (IDDM)

A literature survey and hypothesis is presented in which it is concluded that an intracellular ventromedial hypothalamic (VMH) glucopenia results in a bibrachial response consisting of adenohypophysial release of growth hormone and ACTH as well as sympathetic discharge, both of which act to elevate plasma glucose and remove the VMH glucopenia. This glucopenia may occur as a result of either a deficiency of circulating insulin or alterations in the kinetic properties of the VMH cellular insulin receptor. Two mechanisms for the development of insulin dependent diabetes mellitus (IDDM) are presented: (1) a defect in VMH glucose transport and/or metabolism such that a VMH glucopenia occurs with a subsequent bibrachial response. The result of this is glucose overproduction and a chronic excess glucose stimulus will eventually cause B-cell exhaustion (primary hypothalamic involvement). (2) reduction of the B-cell population by chemical, genetic and/or viral interactions with a consequential insulopenia results in a VMH glucopenia (secondary to a reduced glucose transport into the VMH cells) and causes a bibrachial response. This VMH response may temporarily restore plasma glucose balance but a chronically enhanced counter-regulatory response to maintain this balance will eventually stress the remaining B-cell population and cause further reductions in B-cell numbers (secondary hypothalmic involvement).