Regulatory B Cells-Immunopathological and Prognostic Potential in Humans.

The aim of the following review is to shed light on the putative role of regulatory B cells (Bregs) in various human diseases and highlight their potential prognostic and therapeutic relevance in humans. Regulatory B cells are a heterogeneous group of B lymphocytes capable of suppressing inflammatory immune reactions. In this way, Bregs contribute to the maintenance of tolerance and immune homeostasis by limiting ongoing immune reactions temporally and spatially. Bregs play an important role in attenuating pathological inflammatory reactions that can be associated with transplant rejection, graft-versus-host disease, autoimmune diseases and allergies but also with infectious, neoplastic and metabolic diseases. Early studies of Bregs identified IL-10 as an important functional molecule, so the IL-10-secreting murine B10 cell is still considered a prototype Breg, and IL-10 has long been central to the search for human Breg equivalents. However, over the past two decades, other molecules that may contribute to the immunosuppressive function of Bregs have been discovered, some of which are only present in human Bregs. This expanded arsenal includes several anti-inflammatory cytokines, such as IL-35 and TGF-ß, but also enzymes such as CD39/CD73, granzyme B and IDO as well as cell surface proteins including PD-L1, CD1d and CD25. In summary, the present review illustrates in a concise and comprehensive manner that although human Bregs share common functional immunosuppressive features leading to a prominent role in various human immunpathologies, they are composed of a pool of different B cell types with rather heterogeneous phenotypic and transcriptional properties.

Real-world data suggest effectiveness of the allogeneic mesenchymal stromal cells preparation MSC-FFM in ruxolitinib-refractory acute graft-versus-host disease.

BACKGROUND: Patients with steroid-refractory acute graft-versus-host disease (aGvHD) not tolerating/responding to ruxolitinib (RR-aGvHD) have a dismal prognosis. METHODS: We retrospectively assessed real-world outcomes of RR-aGvHD treated with the random-donor allogeneic MSC preparation MSC-FFM, available via Hospital Exemption in Germany. MSC-FFM is provided as frozen cell dispersion for administration as i.v. infusion immediately after thawing, at a recommended dose of 1-2 million MSCs/kg body weight in 4 once-weekly doses. 156 patients, 33 thereof children, received MSC-FFM; 5% had Grade II, 40% had Grade III, and 54% had Grade IV aGvHD. Median (range) number of prior therapies was 4 (1-10) in adults and 7 (2-11) in children. RESULTS: The safety profile of MSC-FFM was consistent with previous reports for MSC therapies in general and MSC-FFM specifically. The overall response rate at Day 28 was 46% (95% confidence interval [CI] 36-55%) in adults and 64% (45-80%) in children; most responses were durable. Probability of overall survival at 6, 12 and 24 months was 47% (38-56%), 35% (27-44%) and 30% (22-39%) for adults, and 59% (40-74%), 42% (24-58%) and 35% (19-53%) for children, respectively (whole cohort: median OS 5.8 months). CONCLUSION: A recent real-world analysis of outcomes for 64 adult RR-aGvHD patients not treated with MSCs reports survival of 20%, 16% and 10% beyond 6, 12 and 24 months, respectively (median 28 days). Our data thus suggest effectiveness of MSC-FFM in RR-aGvHD.

Cytokine levels associated with favorable clinical outcome in the CAPSID randomized trial of convalescent plasma in patients with severe COVID-19.

Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP). Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models. Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models. Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors.

Control of TLR7-mediated type I IFN signaling in pDCs through CXCR4 engagement-A new target for lupus treatment.

Type I interferons are highly potent cytokines essential for self-protection against tumors and infections. Deregulations of type I interferon signaling are associated with multiple diseases that require novel therapeutic options. Here, we identified the small molecule, IT1t, a previously described CXCR4 ligand, as a highly potent inhibitor of Toll-like receptor 7 (TLR7)-mediated inflammation. IT1t inhibits chemical (R848) and natural (HIV) TLR7-mediated inflammation in purified human plasmacytoid dendritic cells from blood and human tonsils. In a TLR7-dependent lupus-like model, in vivo treatment of mice with IT1t drives drastic reduction of both systemic inflammation and anti-double-stranded DNA autoantibodies and prevents glomerulonephritis. Furthermore, IT1t controls inflammation, including interferon α secretion, in resting and stimulated cells from patients with systemic lupus erythematosus. Our findings highlight a groundbreaking immunoregulatory property of CXCR4 signaling that opens new therapeutic perspectives in inflammatory settings and autoimmune diseases.

ATP promotes immunosuppressive capacities of mesenchymal stromal cells by enhancing the expression of indoleamine dioxygenase.

INTRODUCTION: MSCs are often found within tumors, promote cancer progression and enhance metastasis. MSCs can act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both interfere with effective immune response. Damage associated molecular patterns (DAMPs) including ATP are found within the tumor microenvironment, attract MSCs, and influence their biology. METHODS: Bone marrow derived MSCs were exposed to ATP for 4 days, in the presence of 100 ng IFNγ/mL. Intracellular expression of IDO in MSCs was assessed by FACS. Conditioned media from thus stimulated MSCs was analyzed for kynurenine content and its suppressive effect on lymphocyte proliferation. Apyrase or P2 × 7-receptor antagonist (AZ 11645373) were applied in order to inhibit ATP induced effect on MSCs. RESULTS: We demonstrate, that ATP at concentrations between 0.062 and 0.5 mM increases dose dependently the expression of IDO in MSCs with subsequent increased kynurenine concentrations within the supernatant at about 60%. This effect could be abolished completely in the presence of ATP degrading enzyme (apyrase) or when MSCs were pretreated with a P2 × 7-receptor antagonist (AZ 11645373). Consistently, supernatants from MSCs stimulated with ATP, inhibited lymphocyte proliferation from 65% to 16%. CONCLUSIONS: We characterized ATP as a DAMP family member responsible for necrosis-induced immunomodulation. Given the increased concentration of DAMPs within tumor tissue and the fact that DAMPs can act as chemotattractants to MSCs, our results have implications for therapeutic strategies targeting the tumor microenvironment.

CD4+ T cell-derived IL-21 and deprivation of CD40 signaling favor the in vivo development of granzyme B-expressing regulatory B cells in HIV patients.

IL-21 can induce both plasma cells and regulatory B cells. In this article, we demonstrate that untreated HIV patients display CD4(+) T cells with enhanced IL-21 expression and high in vivo frequencies of regulatory B cells overexpressing the serine protease granzyme B. Granzyme B-expressing regulatory B cells (GraB cells) cells from HIV patients exhibit increased expression of CD5, CD43, CD86, and CD147 but do not produce IL-10. The main functional characteristic of their regulatory activity is direct granzyme B-dependent degradation of the TCR-ζ-chain, resulting in significantly decreased proliferative T cell responses. Although Th cells from HIV patients secrete IL-21 in a Nef-dependent manner, they barely express CD40L. When culturing such IL-21(+)CD40L(-) Th cells with B cells, the former directly induce B cell differentiation into GraB cells. In contrast, the addition of soluble CD40L multimers to T cell/B cell cultures redirects B cell differentiation toward plasma cells, indicating that CD40L determines the direction of IL-21-dependent B cell differentiation. As proof of principle, we confirmed this mechanism in a patient lacking intact CD40 signaling due to a NEMO mutation. The majority of peripheral B cells from this patient were GraB cells and strongly suppressed T cell proliferation. In conclusion, GraB cells represent potent regulatory B cells in humans that are phenotypically and functionally distinct from B10 cells and occur in early HIV infection. GraB cells may contribute significantly to immune dysfunction in HIV patients, and may also explain ineffective Ab responses after vaccination. The use of soluble CD40L multimers may help to improve vaccination responses in HIV patients.

Hepcidin/Ferritin Quotient Helps to Predict Spontaneous Recovery from Iron Loss following Blood Donation.

BACKGROUND: Iron supplementation is generally recommended for blood donors even though there are inter-individual differences in iron homeostasis. METHODS: Ferritin levels of repeat donors were compared with first-time donors, retrospectively. Prospectively, we tested 27 male repeat donors for the following parameters at the day of blood donation as well as 1, 3, 7, 10, and 56 days thereafter: ferritin, hepcidin, transferrin, transferrin receptor, hemoglobin, erythropoietin, reticulocytes, hemoglobin in reticulocyte, twisted gastrulation protein homolog 1, and growth differentiation factor-15. RESULTS: 56 days after blood donation, donors' average ferritin dropped to 55% (range 30-100%) compared to the initial value. Of all tested parameters hepcidin showed the highest and most significant changes beginning 1 day after donation and lasting for the whole period of 56 days. Along with ferritin, there was a high variation in hepcidin levels indicating inter-individual differences in hepcidin response to iron loss. Donors with a hepcidin/ferritin quotient < 0.3 regained 60% of their initial ferritin after 56 days, while those with a quotient ≥ 0.3 reached less than 50%. CONCLUSION: As hepcidin appears to integrate erythropoietic and iron-loading signals, clinical measurement of hepcidin (together with the hepcidin-ferritin ratio) may become a useful indicator of erythropoiesis and iron kinetics.

S100A4 and uric acid promote mesenchymal stromal cell induction of IL-10+/IDO+ lymphocytes.

Simple stress or necrotic cell death with subsequent release of damage-associated molecular patterns (DAMPs) is a characteristic feature of most advanced tumors. DAMPs within the tumor microenvironment stimulate tumor-associated cells, including dendritic cells and mesenchymal stromal cells (MSCs). The presence of tumor-infiltrating MSCs is associated with tumor progression and metastasis. Oxidized necrotic material loses its stimulatory capacity for MSCs. As a DAMP, S100A4 is sensitive to oxidation whereas uric acid (UA) acts primarily as an antioxidant. We tested these two biologic moieties separately and in combination for their activity on MSCs. Similar to necrotic tumor material, S100A4 and UA both dose-dependently induced chemotaxis of MSCs with synergistic effects when combined. Substituting for UA, alternative antioxidants (vitamin C, DTT, and N-acetylcysteine) also enhanced the chemotactic activity of S100A4 in a synergistic manner. This emphasizes the reducing potential of UA being, at least in part, responsible for the observed synergy. With regard to MSC proliferation, both S100A4 and UA inhibited MSCs without altering survival or inducing differentiation toward adipo-, osteo-, or chondrocytes. In the presence of S100A4 or UA, MSCs gained an immunosuppressive capability and stably induced IL-10- and IDO-expressing lymphocytes that maintained their phenotype following proliferation. We have thus demonstrated that both S100A4 and UA act as DAMPs and, as such, may play a critical role in promoting some aspects of MSC-associated immunoregulation. Our findings have implications for therapeutic approaches targeting the tumor microenvironment and addressing the immunosuppressive nature of unscheduled cell death within the tumor microenvironment.

B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21.

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.

Interleukin 21-induced granzyme B-expressing B cells infiltrate tumors and regulate T cells.

The pathogenic impact of tumor-infiltrating B cells is unresolved at present, however, some studies suggest that they may have immune regulatory potential. Here, we report that the microenvironment of various solid tumors includes B cells that express granzyme B (GrB, GZMB), where these B cells can be found adjacent to interleukin (IL)-21-secreting regulatory T cells (Treg) that contribute to immune tolerance of tumor antigens. Because Tregs and plasmacytoid dendritic cells are known to modulate T-effector cells by a GrB-dependent mechanism, we hypothesized that a similar process may operate to modulate regulatory B cells (Breg). IL-21 induced outgrowth of B cells expressing high levels of GrB, which thereby limited T-cell proliferation by a GrB-dependent degradation of the T-cell receptor ζ-chain. Mechanistic investigations into how IL-21 induced GrB expression in B cells to confer Breg function revealed a CD19(+)CD38(+)CD1d(+)IgM(+)CD147(+) expression signature, along with expression of additional key regulatory molecules including IL-10, CD25, and indoleamine-2,3-dioxygenase. Notably, induction of GrB by IL-21 integrated signals mediated by surface immunoglobulin M (B-cell receptor) and Toll-like receptors, each of which were enhanced with expression of the B-cell marker CD5. Our findings show for the first time that IL-21 induces GrB(+) human Bregs. They also establish the existence of human B cells with a regulatory phenotype in solid tumor infiltrates, where they may contribute to the suppression of antitumor immune responses. Together, these findings may stimulate novel diagnostic and cell therapeutic approaches to better manage human cancer as well as autoimmune and graft-versus-host pathologies.

Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.

B cells are generally believed to operate as producers of high affinity antibodies to defend the body against microorganisms, whereas cellular cytotoxicity is considered as an exclusive prerogative of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). In conflict with this dogma, recent studies have demonstrated that the combination of interleukin-21 (IL-21) and B-cell receptor (BCR) stimulation enables B cells to produce and secrete the active form of the cytotoxic serine protease granzyme B (GrB). Although the production of GrB by B cells is not accompanied by that of perforin as in the case of many other GrB-secreting cells, recent findings suggest GrB secretion by B cells may play a significant role in early antiviral immune responses, in the regulation of autoimmune responses, and in cancer immunosurveillance. Here, we discuss in detail how GrB-secreting B cells may influence a variety of immune processes. A better understanding of the role that GrB-secreting B cells are playing in the immune system may allow for the development and improvement of novel immunotherapeutic approaches against infectious, autoimmune and malignant diseases.

Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help.

Recently, CD4(+) T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL-)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4(+) T cell-derived IL-21 induces differentiation of B cells into granzyme B (GzmB)-secreting cytotoxic cells. Using fluorescence-activated cell sorting (FACS) analysis, ELISpot and confocal microscopy, we demonstrate that CD4(+) T cells, activated via their T-cell receptor without co-stimulation, can produce IL-21, but do not express CD40L and rapidly induce GzmB in co-cultured B cells in an IL-21 receptor-dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40L suppresses IL-21-induced GzmB induction in B cells in a dose-dependent manner. Surprisingly, although GzmB-secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL-2 instead of IL-21 or when target cells were cultured with IL-21 alone. Our findings suggest GzmB(+) cytotoxic B cells may have a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen-specific cytotoxic T cells are on the spot. CD40 ligand determines whether IL-21 induces differentiation of B cells into plasma cells or into granzyme B-secreting cytotoxic cells.

Approaches to the pharmacological modulation of plasmacytoid dendritic cells.

Human plasmacytoid dendritic cells (pDC) are crucial for the modulation of adaptive immune responses in the course of neoplastic, viral and autoimmune diseases. In several of these disorders deregulated pDC-derived interferon-α (IFN-α), a key cytokine produced by pDC, plays a central role. Apart from IFN-α, pDC can produce a variety of other mediators, which are involved in immunological cross-talk. The most recently discovered are the cytotoxic serine protease granzyme B (GrB) and indoleamine 2,3-dioxygenase, which have been described to be involved in the suppression of effector T cell responses. Here we review the regulation of pDC function by a variety of immunomodulatory agents, which may be developed as future candidates for the therapy of a variety of diseases. Moreover, we introduce the novel concept of enhancing immune responses after vaccination in poor responders by increasing pDC-derived IFN-α and simultaneously inhibiting pDC-derived GrB secretion. Finally we discuss potential approaches to abrogate pDC-mediated tolerance induction against tumors and viral infections.

CD5+ B cells from individuals with systemic lupus erythematosus express granzyme B.

Recently, we reported that IL-21 induces granzyme B (GzmB) and GzmB-dependent apoptosis in malignant CD5(+) B cells from patients with chronic lymphocytic leukemia. Several autoimmune diseases (AD) are associated with enhanced frequencies of CD5(+) B cells. Since AD are also associated with elevated IL-21 and GzmB levels, we postulated a link between CD5(+) B cells, IL-21 and GzmB. Here, we demonstrate that IL-21 and GzmB serum levels are highly correlated in subjects with systemic lupus erythematosus (SLE) and that freshly isolated CD5(+) SLE B cells constitutively express GzmB. IL-21 directly induced GzmB expression and secretion by CD5(+) B cells from several AD and from cord blood in vitro, and the simultaneous presence of BCR stimulation strongly enhanced this process. Furthermore, IL-21 suppressed both viability and expansion of CD5(+) B cells from SLE individuals. In summary, our study may explain the elevated levels of IL-21 and GzmB in SLE and other AD. Moreover, our data suggest that IL-21 may have disease-modifying characteristics by inducing GzmB in CD5(+) B cells and by suppressing their expansion. Our results provide the rationale for further evaluation of the therapeutic potential of IL-21 in certain AD such as SLE.

Granzyme B produced by human plasmacytoid dendritic cells suppresses T-cell expansion.

Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB(+) pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.

CpG oligodeoxynucleotides as immunotherapy in cancer.

Preclinical and early clinical trials indicate synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG ODN) have potent immunostimulatory effects and can enhance the anti-cancer activity of a variety of cancer treatments. Synergy between CpG ODN and monoclonal antibodies has been noted in various preclinical models. Early clinical trials indicate CpG ODN and monoclonal antibodies can be administered safely together. Preclinical models indicate CpG ODN can enhance the anti-tumor activity of both chemotherapy and radiation therapy. Thus, one possible approach to the use of CpG ODN was to use it in combination with cytotoxic chemotherapy with the goal of enhancing presentation of tumor antigen from dying cancer cells. Promising results in a randomized phase II trial in patients with non-small cell lung cancer led to initiation of two large randomized phase III trials comparing CpG ODN plus chemotherapy to chemotherapy alone. Unfortunately, interim analysis of these trials indicated CpG ODN was unlikely to enhance efficacy of chemotherapy, and they were stopped. CpG ODN also holds promise as a component of cancer vaccines including those composed of protein antigen, peptides, whole tumor cells, and antigen-pulsed dendritic cells. Finally, CpG ODN has been combined with a variety of cytokines to enhance NK activation, promote development of an active anti-tumor immune response or induce apoptosis of malignant cells that express the TLR9 receptor. Overall, both preclinical and early clinical trials suggest CpG ODN may be a valuable component of a variety of approaches to cancer therapy. However, clinical development of this recently discovered, novel class of immunostimulatory agents is just beginning, and we still have much to learn about the optimal approach to their use, and their potential.

Human plasmacytoid dendritic cell function: inhibition of IFN-alpha secretion and modulation of immune phenotype by vasoactive intestinal peptide.

Plasmacytoid dendritic cells (PDC) are considered the main sentinels against viral infections and play a major role in immune tolerance. Vasoactive intestinal peptide (VIP) is a potent immunomodulator, whose role in PDC function is unknown. The present study was designed to investigate whether human PDC express VIP receptors and whether VIP has immunological effects on PDC. Using real-time RT-PCR and immunofluorescence, we demonstrated that VIP receptors VPAC1 and VPAC2 are expressed on PDC. After culturing PDC with VIP and CpG oligodeoxynucleotides for 48 h, expression of surface molecules with significance for PDC-T cell interactions as well as IFN-alpha secretion were quantified using FACS analysis and ELISA, respectively. For functional assays, CFSE-stained CD4+ T cells were coincubated with differentially treated PDC. T cell proliferation and production of various cytokines were determined by FACS analysis and ELISA. VIP enhanced PDC expression of CD86, MHC II, and CCR7. In contrast, VIP inhibited PDC secretion of IFN-alpha and expression of Neuropilin-1 and MHC I. The potential of CpG oligodeoxynucleotide-activated PDC to induce proliferation of allogeneic CD4(+) T cells was impaired when VIP was present during activation. Furthermore, pretreatment of PDC with VIP resulted in a decrease of the IFN-gamma:IL-4 ratio in cocultured T cells, suggesting a modulation of the immune response toward Th2. Taken together, these results strongly suggest that VIP regulates the immunological function of human PDC. VIP may thus be involved in the modulation of immune responses to viral infections as well as in the maintenance of immune tolerance.

B-chronic lymphocytic leukemia cells and other B cells can produce granzyme B and gain cytotoxic potential after interleukin-21-based activation.

B cells currently are not viewed as being capable of producing granzyme B or being cytotoxic. We found that B-chronic lymphocytic leukemia (B-CLL) cells treated with interleukin-21 (IL-21) produce low levels of granzyme B. The addition of either CpG oligodeoxynucleotide (ODN) or anti-B-cell-receptor antibody (anti-BCR) to IL-21 results in enhanced production of functional granzyme B by B-CLL cells. B-CLL cells treated with IL-21 and CpG ODN undergo apoptosis and are able to induce apoptosis of untreated bystander B-CLL cells. This effect can be inhibited by anti-granzyme B antibody. Benign human B cells, Epstein-Barr virus (EBV)-transformed lymphoblasts, and many standard lymphoma cell lines produce high levels of granzyme B in response to IL-21 and anti-BCR. Our results suggest that the ability to induce production of functional granzyme B by B cells could open new approaches to the therapy of B-CLL and other B-cell malignancies. Our findings also have significant implications for our understanding of the role of B cells for immune regulation and for a variety of immune phenomena, including cancer immunity, autoimmunity, and infectious immunity.