Inflammation Causes Resistance to Anti-CD20-Mediated B Cell Depletion.

B cells play a central role in antibody-mediated rejection and certain autoimmune diseases. However, B cell-targeted therapy such as anti-CD20 B cell-depleting antibody (aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B cell reconstitution leading to aCD20 depletion resistance could account for these discrepancies. Using a transplantation model, we found that antigen-independent inflammation, likely through toll-like receptor (TLR) signaling, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics (PK) and pharmacodynamics (PD) of aCD20 therapy by shortening drug half-life and accelerating the reconstitution of the peripheral B cell pool by bone marrow-derived B cell precursors. IVIG (intravenous immunoglobulin) coadministration also shortened aCD20 drug half-life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell-dependent, antibody-independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions. The data also highlight the disconnect between B cell depletion on peripheral blood compared to secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20 and the relevance of redosing of aCD20 for effective B cell depletion in alloimmunity.

Depot-specific differences in inflammatory mediators and a role for NK cells and IFN-gamma in inflammation in human adipose tissue.

BACKGROUND: Adipose tissue is a primary in vivo site of inflammation in obesity. Excess visceral adipose tissue (VAT), when compared to subcutaneous adipose tissue (SAT), imparts an increased risk of obesity-related comorbidities and mortality, and exhibits differences in inflammation. Defining depot-specific differences in inflammatory function may reveal underlying mechanisms of adipose-tissue-based inflammation. METHODS: Stromovascular cell fractions (SVFs) from VAT and SAT from obese humans undergoing bariatric surgery were studied in an in vitro culture system with transcriptional profiling, flow cytometric phenotyping, enzyme-linked immunosorbent assay and intracellular cytokine staining. RESULTS: Transcriptional profiling of SVF revealed differences in inflammatory transcript levels in VAT relative to SAT, including elevated interferon-gamma (IFN-gamma) transcript levels. VAT demonstrated a broad leukocytosis relative to SAT that included macrophages, T cells and natural killer (NK) cells. IFN-gamma induced a proinflammatory cytokine expression pattern in SVF and adipose tissue macrophages (ATM). NK cells, which constitutively expressed IFN-gamma, were present at higher frequency in VAT relative to SAT. Both T and NK cells from SVF expressed IFN-gamma on activation, which was associated with tumor necrosis factor-alpha expression in macrophages. CONCLUSION: These data suggest involvement of NK cells and IFN-gamma in regulating ATM phenotype and function in human obesity and a potential mechanism for the adverse physiologic effects of VAT.

Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR.

Unlike B cells, T cells lack the capacity to improve the affinity of their antigen receptors by somatic mutation. It is, therefore, believed that optimization of cellular immunity is mediated almost exclusively through selective expansion of T cells bearing receptors with the highest affinity for antigen. We show here that T cell responsiveness to peptide (termed "functional avidity") increased>50-fold during the early stages of viral infection. This indicated that T cells, like B cells, undergo extensive functional maturation in vivo. However, in contrast to B cells, maturation of the T cell response can occur without any appreciable change in T cell receptor affinity.

Two overlapping subdominant epitopes identified by DNA immunization induce protective CD8(+) T-cell populations with differing cytolytic activities.

Subdominant CD8(+) T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by L(d) and the other presented by K(d). This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8(+) T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8(+) response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the "dominant" vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the "subdominant" vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8(+) T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysis(low)-cytokine(competent) phenotype associated with most memory CD8(+) T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8(+) T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8(+) T cells with distinct functional attributes.

Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8+ T cells during picornavirus infection.

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization.

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.

Clinical implications of dysregulated cytokine production.

Cytokines are soluble proteins that are produced and secreted as part of the immune response to a variety of tissue insults including infection, cancer, and autoimmunity. Most cytokines are secreted by cells of the immune system, but some (for example, type I interferons) are released from "nonimmunological" cells such as fibroblasts and epithelial cells. Cytokines have pleiotropic effects, acting on many somatic cell types to modulate the host's immune response. For the most part, cytokines exert their antimicrobial actions locally---they are secreted by cells in the area of infection, and their effects are restricted to neighboring cells. While many of their local effects benefit the host, cytokines are soluble molecules that may act systemically and are often responsible for many of the symptoms of infection (e.g., headache, fever, myalgia). In high concentrations they can be toxic, or even lethal. Human clinical trials involving the systemic injection of purified cytokines such as interleukins 2 and 12 and tumor necrosis factor alpha provide compelling evidence for the toxicity of these molecules. Likewise, studies of septic shock syndrome demonstrate how overproduction/aberrant production of inflammatory cytokines can lead to rapid mortality. The host may attempt to counter high cytokine levels by releasing soluble cytokine receptors (sCR) or by synthesizing high-affinity anti-cytokine antibodies (acAb), and these natural responses have spawned great interest as potential therapeutic approaches for alleviating cytokine-mediated disease. However, recent studies indicate that these in vivo interactions are much more complex than previously realized; administration of sCR or acAb may either inhibit or (paradoxically) enhance cytokine activity. An alternative therapeutic approach is to intervene at the source of cytokine production. T cells initiate cytokine production only upon antigen contact and terminate synthesis almost immediately after this contact is broken. Thus T cells secrete cytokines specifically at sites of infection and do not continuously produce these potentially toxic molecules while migrating through uninfected tissues or the bloodstream. By learning more about the molecular mechanisms involved with on/off regulation of cytokine production we may be able to develop novel therapeutic drugs to protect against cytokine-mediated immunopathology. This review discusses the regulation of cytokine function by sCR and acAb and compares this to the regulatory mechanisms that are associated with antigen-specific cytokine release by T cells.

NK markers are expressed on a high percentage of virus-specific CD8+ and CD4+ T cells.

NK cells have been phenotypically defined by the expression of specific markers such as NK1.1, DX5, and asialo-GM1 (ASGM1). In addition to NK cells, a small population of CD3+ T cells has been shown to express these markers, and a unique subpopulation of NK1. 1+CD3+ T cells that expresses an invariant TCR has been named "NKT cells." Here, we describe NK marker expression on a broad spectrum of MHC class I- and MHC class II-restricted T cells that are induced after acute viral infection. From 5 to >500 days post lymphocytic choriomeningitis virus (LCMV) infection, more than 90% of virus-specific CD8+ and CD4+ T cells coexpress one or more of these three prototypical NK markers. Furthermore, in vivo depletion of NK cells with anti-ASGM1 Ab resulted in the removal of 90% of virus-specific CD8+ T cells and 50-80% of virus-specific CD4+ T cells. This indicates that studies using in vivo depletion to determine the role of NK cells in immune defense could potentially be misinterpreted because of the unintended depletion of Ag-specific T cells. These results demonstrate that NK Ags are widely expressed on the majority of virus-specific T cells and indicate that the NK and T cell lineages may not be as distinct as previously believed. Moreover, the current nomenclature defining NKT cells will require comprehensive modification to include Ag-specific CD8+ and CD4+ T cells that express prototypical NK Ags.

Activated and memory CD8+ T cells can be distinguished by their cytokine profiles and phenotypic markers.

Dissecting the mechanisms of T cell-mediated immunity requires the identification of functional characteristics and surface markers that distinguish between activated and memory T lymphocytes. In this study, we compared the rates of cytokine production by virus-specific primary and memory CD8+ T cells directly ex vivo. Ag-specific IFN-gamma and TNF-alpha production by both primary and long-term memory T cells was observed in

Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells.

CD8-positive T cells protect the body against viral pathogens by two important mechanisms: production of antiviral cytokines and lysis of infected cells. Cytokine production can have both local and systemic consequences, whereas cytolytic activity is limited to infected cells that are in direct contact with T cells. Here we analyse activated CD8-positive T cells from mice infected with lymphocytic choriomeningitis virus and find that cytokines are not produced ex vivo in the absence of peptide stimulation, but that they are rapidly generated after T cells encounter viral peptides bound to the major histocompatibility complex. Remarkably, cytokine production ceases immediately upon dissociation of the T cells from their targets and resumes when antigenic contact is restored. In contrast to the 'on/off/on' cycling of cytokines, the pore-forming cytotoxic protein perforin is constitutively maintained. Our results indicate that there is differential expression of effector molecules according to whether the antiviral product is secreted (like cytokines) or stored inside the cell (like perforin). The ability to turn cytokines on and off while maintaining intracellular stores of perforin shows the versatility of the cellular immune response and provides a mechanism for maintaining effective immune surveillance while reducing systemic immunopathology.

Long-lived plasma cells: a mechanism for maintaining persistent antibody production.

Current models suggest that continuous antigenic stimulation of memory B cells is required to maintain long-term antibody production. In view of recent developments concerning plasma cell longevity, a new model is described that incorporates the important role of long-lived plasma cells in sustaining persistent antibody responses.

CD4+ and CD8+ T cell interactions in IFN-gamma and IL-4 responses to viral infections: requirements for IL-2.

Cytokine responses to lymphocytic choriomeningitis virus infections were evaluated, and CD8+ T cell, CD4+ T cell, and IL-2 contributions delineated. In immunocompetent mice, lymphocytic choriomeningitis virus induced both IFN-gamma and IL-4 as well as IL-2. Experiments in mice either beta2-microglobulin-deficient, lacking MHC class I molecules and CD8+ T cells, or A beta(b)-deficient, lacking MHC class II molecules and CD4+ T cells, demonstrated that mixtures of T cell responses were required for optimal ex vivo cytokine productions. Intracellular cytokine expression analyses of cells from immunocompetent and immunodeficient mice showed that CD8+ T cells were predominant IFN-gamma producers, and that expansion of CD8+ T cells primed to make IFN-gamma was independent of CD4+ T cells in vivo. Studies in IL-2-deficient mice demonstrated that this cytokine promoted IFN-gamma and IL-4 responses, and ex vivo experiments showed that exogenous IL-2 was required to maintain high-level IFN-gamma production by in vivo-primed CD8+ T cells. Conditions associated with cytokine decreases were accompanied by reduced detectable plasma Ab responses. The results indicate that, although IL-2-dependent CD8+ T cell proliferation does not require endogenous CD4+ T cells, IL-2 production by the CD4+ T cells may promote continued cytokine release from activated CD8+ T cells. By defining these critical steps in cellular and cytokine interactions for shaping endogenous immune responses, the studies advance understanding of the unique conditions regulating CD8+ T cell responses to viral challenges.

Humoral immunity due to long-lived plasma cells.

Conventional models suggest that long-term antibody responses are maintained by the continuous differentiation of memory B cells into antibody-secreting plasma cells. This is based on the notion that plasma cells are short-lived and need to be continually replenished by memory B cells. We examined the issue of plasma cell longevity by following the persistence of LCMV-specific antibody and plasma cell numbers after in vivo depletion of memory B cells and by adoptive transfer of virus-specific plasma cells into naive mice. The results show that a substantial fraction of plasma cells can survive and continue to secrete antibody for extended periods of time (>1 year) in the absence of any detectable memory B cells. This study documents the existence of long-lived plasma cells and demonstrates a new mechanism by which humoral immunity is maintained.

Bone marrow contains virus-specific cytotoxic T lymphocytes.

Immunizing bone marrow donors prior to bone marrow transplant (BMT) has the potential for adoptively transferring specific immunity against opportunistic pathogens. Studies have shown that long-term antibody production occurs in the bone marrow and that specific humoral immunity may be transferred from donor to recipient following BMT. However, the magnitude and duration of T-cell memory in the bone marrow compartment has not been adequately investigated. In this study, virus-specific CD8+ T-cell responses in the bone marrow were compared with those observed in the spleen of mice acutely infected with lymphocytic choriomeningitis virus (LCMV). During the acute stages of infection, most CD8+ T cells in the spleen and bone marrow showed upregulated surface expression of the activation/memory marker, LFA-1 (LFA-1[hi]). After clearing LCMV infection, the antiviral immune response subsided to homeostatic levels and the ratio of CD8+/LFA-1(hi) to CD8+/LFA-1(lo) T cells in the spleen and bone marrow of LCMV immune mice returned to the value observed in naive mice. Virus-specific ex vivo effector cytotoxic T-lymphocyte (CTL) responses could be identified in both spleen and bone marrow compartments at 8 days postinfection. LCMV-specific CTL precursor (CTLp) frequencies peaked in the bone marrow at 8 days postinfection and averaged one in 200 to one in 650 CD8+ T cells, a frequency similar to that observed in the spleen. After clearing the acute infection, potent LCMV-specific CTL memory responses could be demonstrated in the bone marrow for at least 325 days postinfection, indicating long-term persistence of antiviral T cells at this site. Adoptive transfer of LCMV-immune bone marrow into severe combined immunodeficiency (SCID) mice provided protection against viral challenge, whereas SCID mice that received naive bone marrow became chronically infected upon challenge with LCMV. These results indicate that after acute viral infection, virus-specific memory T cells can be found in the bone marrow compartment and are maintained for an extended period, and when adoptively transferred into an immunodeficient host, they are capable of conferring protection against chronic viral infection.

CD40 ligand-deficient mice generate a normal primary cytotoxic T-lymphocyte response but a defective humoral response to a viral infection.

CD40 ligand is expressed on activated T cells and interacts with CD40 on B cells and monocytes. It is not known what role CD40 ligand plays in the generation of immune responses to viral infection. To address this issue, we examined virus-specific T- and B-cell responses in CD40 ligand-deficient (CD40L-/-) mice following infection with lymphocytic choriomeningitis virus (LCMV). We found that primary anti-LCMV specific antibody responses were severely impaired in CD40L-/- mice, with the defect being most striking for antibody of the immunoglobulin G1 (IgG1) isotype. Interestingly, low levels of LCMV-specific antibodies of the IgG2a, IgG2b, and IgG3 isotypes were made in the CD40L-/- mice, showing that IgG1 responses are totally dependent on CD40L but that at least some IgG2a, IgG2b, and IgG3 responses can be CD40L independent. However, unlike CD40L+/+ mice, CD40L-/- mice were unable to sustain virus-specific antibody responses and showed a gradual decline in serum antibody levels over time. The CD40L-/- mice were also deficient in the generation of memory B cells. In contrast to the severely impaired humoral responses, CD40L-/- mice generated potent virus-specific CD8+ cytotoxic T-lymphocyte responses after LCMV infection and were able to clear the virus. These results show that CD40L does not play a role in generating primary virus-specific CD8+ cytotoxic T-lymphocyte responses but does affect the primary antibody response and the generation of memory B cells.

Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay.

Many acute viral infections induce long-term, sometimes even life-long humoral immunity. To characterize this immune response, accurate quantitation of memory B cells and plasma cells is essential. Plasma cells can be quantitated directly ex vivo by virtue of their ability to spontaneously secrete antibody. Memory B cells on the other hand, do not spontaneously secrete antibody but require antigenic stimulation in order to proliferate and differentiate into antibody secreting cells (ASC). In this study, an ELISPOT-based limiting dilution assay (LDA) was developed for quantitating virus-specific B cell memory following acute lymphocytic choriomeningitis virus (LCMV) infection of adult mice. Antiviral memory B cell precursor (MBCp) frequencies were calculated from in vitro stimulated cultures using either a conventional ELISA-based LDA to measure accumulated virus-specific antibody in the culture medium or a new ELISPOT-based LDA that identifies the antibody-secreting daughter cells directly. In terms of sensitivity, the ELISPOT-based LDA and the ELISA-based LDA both calculated LCMV-specific MBCp frequencies to be approximately 1/2 x 10(4) spleen cells. However, compared to the 12 days of in vitro stimulation required to estimate MBCp frequencies by the ELISA-based LDA, the ELISPOT-based LDA required only 3-6 days of stimulation to quantitate MBCp frequencies. If cell division was blocked by gamma-irradiation or treatment with mitomycin C, the MBCp frequency dropped below detection (< 1 MBCp/10(6) cells), indicating that virus-specific B cells quantitated by this assay must both proliferate and differentiate into antibody secreting cells in order to be detected. Naive, uninfected mice did not have LCMV-specific memory B cells, demonstrating that only in vivo-generated antiviral B cells from LCMV-immune mice were detected by this assay. chi 2 analysis of the ELISPOT-based LDA showed that the MBCp frequency data fit a linear regression model (p = 0.0137), indicating single-hit kinetics in which only one cell type was limiting. These results indicate that the ELISPOT-based LDA provides a rapid and statistically accurate method to quantitate virus-specific B cells.