Effects of rituximab and dexamethasone on regulatory and proinflammatory B-cell subsets in patients with primary immune thrombocytopenia.

OBJECTIVE: To investigate the cytokine production and surface marker composition of B cells in adult patients with newly diagnosed primary immune thrombocytopenia (ITP) before and 12 months after treatment with rituximab + dexamethasone (RTX+DXM) or dexamethasone (DXM). METHODS: Peripheral blood mononuclear cells were isolated from nine patients treated with RTX+DXM, seven patients treated with DXM, and seven healthy donors. Expression of the cell-surface markers CD5, CD27, CD25, and CD19, and intracellular content of IL-6 and IL-10 were measured by flow cytometry. RESULTS: PBMCs from ITP patients at baseline contained a lower proportion of IL-10+ B cells (P < .01) and IL-6+ B cells (P < .01) than healthy controls. All patients responded to therapy and levels were normalized at 12 months. The proportion of CD5+ B cells increased (P < .01) and CD27+ memory B cells decreased (P < .05) 12 months after treatment with RTX+DXM compared to baseline, with an inverse correlation between platelet numbers and the proportion of CD27+ B cells (R = -0.71; P < .05). CONCLUSION: Both treatment regimens normalized the frequencies of cytokine-producing B cells. The additional increase in CD5+ B cells after RTX+DXM is compatible with induction of Bregs.

Interferon-α induces marked alterations in circulating regulatory T cells, NK cell subsets, and dendritic cells in patients with JAK2V617F-positive essential thrombocythemia and polycythemia vera.

Long-term therapy with IFN-α2 is associated with sustained major molecular remissions in JAK2-positive ET and PV. The efficacy of IFN-α2 may be partly mediated by modulation of immune cells, which was investigated in twenty patients with ET (n = 6) and PV (n = 14). The frequency of CD4(+) CD25(+) Foxp3(+) T cells was significantly increased during IFN-α2 treatment in all patients (P < 0.0001). A significant expansion of the CD56(bright) NK cells (P = 0.0002) and a concomitant decrease in the frequency of CD56(dim) NK cells (P < 0.0001) were also detected. Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were studied in nine patients, and decreased frequencies of both cell types were observed during the course of treatment. On both mDCs and pDCs, HLA-ABC expression was upregulated (P = 0.003), but decreasing expression levels of HLA-DR was detected on mDCs. The expression of CD40 (P = 0.002), CD83 (P = 0.03), and CD86 (P = 0.01) increased, but was confined to pDCs. Furthermore, PD-L1 expression was reduced on mDC (P = 0.003) and increased on pDCs (P = 0.02). No significant correlations were found between the changes in immune cells and hematological or molecular responses achieved in our cohort of patients. So forth, it remains to be revealed whether the profound changes in circulating immune cells contribute to the beneficial effects of long-term IFN-α2 treatment in some patients.

Expansion of circulating CD56bright natural killer cells in patients with JAK2-positive chronic myeloproliferative neoplasms during treatment with interferon-α.

In recent years, major molecular remissions have been observed in patients with JAK2-positive chronic myeloproliferative neoplasms (MPNs) after therapy with IFN-α. IFN-α is known to have altering effects on immune cells involved in immune surveillance and might consequently enhance anti-tumor immune response against the JAK2-mutated clone. The objective of this study was to investigate circulating levels and phenotype of natural killer cells in 29 JAK2-positive MPN patients during IFN-α treatment. Furthermore, functional studies of NK cells upon target-cell recognition and cytokine stimulation were performed. The CD56(bright) and CD56(dim) NK cell subtypes display different properties in terms of cytokine production and cytotoxicity, respectively. Our results show a significant increase in the proportion of CD56(bright) NK cells and a decreasing CD56(dim) population during treatment with IFN-α compared to patients that are untreated, treated with hydroxyurea and healthy controls, P < 0.0001. Furthermore, an overall increase in cytokine-dependent (IL-12 and IL-15) IFN-γ expression by CD56(dim) NK cells during IFN-α treatment was observed. In contrast, our data indicate a compromised NK cell response to target-cell recognition during treatment with IFN-α in four patients. We also report low levels of circulating NK cells in untreated patients compared to healthy donors, patients treated with hydroxyurea and IFN-α, P = 0.02. Based on our findings, one might speculate whether treatment with IFN-α skews the human NK population toward a helper type that may assist in CD8(+) T cell priming in lymphoid tissues at the expense of their immediate cytotoxic functions in peripheral blood and tissues.

Increase in circulating CD4⁺CD25⁺Foxp3⁺ T cells in patients with Philadelphia-negative chronic myeloproliferative neoplasms during treatment with IFN-α.

Recent reports have described complete or major molecular remission in patients with polycythemia vera after long-term treatment with the immunomodulatory agent IFN-α2. Accordingly, there are reasons to believe that the immune system is a key player in eradicating the JAK2 mutated clone in these patients. Foxp3(+) regulatory T cells play a pivotal role in maintaining immune homeostasis and, importantly, preventing immune reactivity to self-antigens; however, their suppressive activity can compromise an effective antitumor immune response, and high frequencies of regulatory T cells in peripheral blood have been reported in both hematologic and solid cancers. We have analyzed the number, phenotype, and function of circulating CD4(+)CD25(+)Foxp3(+) T cells in patients with chronic myeloproliferative neoplasms. Surprisingly, we found a marked expansion of this subset of lymphocytes in patients treated with IFN-α2 (13.0%; 95% confidence interval [CI] 10.8% to 15.2%) compared with healthy donors (6.1%; 95% CI 4.9% to 7.2%), patients with untreated chronic myeloproliferative neoplasms (6.9%; 95% CI 5.8% to 7.4%), or patients treated with hydroxyurea (5.8%; 95% CI 4.3% to 7.4%; P < .0001).

Generation of autologous tumor-specific T cells for adoptive transfer based on vaccination, in vitro restimulation and CD3/CD28 dynabead-induced T cell expansion.

Adoptive cell transfer (ACT) of in vitro expanded autologous tumor-infiltrating lymphocytes (TIL) has been shown to exert therapeutic efficacy in melanoma patients. We aimed to develop an ACT protocol based on tumor-specific T cells isolated from peripheral blood and in vitro expanded by Dynabeads® ClinExVivo™CD3/CD28. We show here that the addition of an in vitro restimulation step with relevant peptides prior to bead expansion dramatically increased the proportion of tumor-specific T cells in PBMC-cultures. Importantly, peptide-pulsed dendritic cells (DCs) as well as allogeneic tumor lysate-pulsed DCs from the DC vaccine preparation could be used with comparable efficiency to peptides for in vitro restimulation, to increase the tumor-specific T-cell response. Furthermore, we tested the use of different ratios and different types of Dynabeads® CD3/CD28 and CD3/CD28/CD137 T-cell expander, for optimized expansion of tumor-specific T cells. A ratio of 1:3 of Dynabeads® CD3/CD28 T-cell expander to T cells resulted in the maximum number of tumor-specific T cells. The addition of CD137 did not improve functionality or fold expansion. Both T-cell expansion systems could generate tumor-specific T cells that were both cytotoxic and effective cytokine producers upon antigen recognition. Dynabeads®-expanded T-cell cultures shows phenotypical characteristics of memory T cells with potential to migrate and expand in vivo. In addition, they possess longer telomeres compared to TIL cultures. Taken together, we demonstrate that in vitro restimulation of tumor-specific T cells prior to bead expansion is necessary to achieve high numbers of tumor-specific T cells. This is effective and easily applicable in combination with DC vaccination, by use of vaccine-generated DCs, either pulsed with peptide or tumor-lysate.

Influenza virus-induced dendritic cell maturation is associated with the induction of strong T cell immunity to a coadministered, normally nonimmunogenic protein.

We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-gamma, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-gamma. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2-3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently.

In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.

The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

Ruxolitinib treatment reduces monocytic superoxide radical formation without affecting hydrogen peroxide formation or systemic oxidative nucleoside damage in myelofibrosis.

The role of excess reactive oxygen species (ROS) with consequent DNA/RNA damage is now recognized as a hallmark of cancer. In JAK2V617F mutated myeloproliferative neoplasms, ROS have been suggested to be important factors in disease initiation and progression. Ruxolitinib is the most widely used drug for myelofibrosis, because it improves symptom-score. However, both the anti-clonal potential and improvement in overall survival are limited. We investigated the impact of ruxolitinib on formation of superoxide radical and hydrogen peroxide by monocytes in sequentially acquired blood samples from patients with myelofibrosis. We also investigated the impact on RNA and DNA damage by measuring urinary excretion of 8-oxo-Guo and 8-oxo-d-Guo. The formation of superoxide by monocytes was reduced significantly during ruxolitinib therapy, but no impact on the formation of hydrogen peroxide by monocytes or the systemic amount of oxidatively damaged RNA or DNA could be demonstrated. We conclude that ruxolitinib holds little anti-oxidative potential.

Myelin Basic Protein-Induced Production of Tumor Necrosis Factor-α and Interleukin-6, and Presentation of the Immunodominant Peptide MBP85-99 by B Cells from Patients with Relapsing-Remitting Multiple Sclerosis.

B cells are involved in driving relapsing-remitting multiple sclerosis (RRMS), as demonstrated by the positive effect of therapeutic B-cell depletion. Aside from producing antibodies, B cells are efficient antigen-presenting and cytokine-secreting cells. Diverse polyclonal stimuli have been used to study cytokine production by B cells, but here we used the physiologically relevant self-antigen myelin basic protein (MBP) to stimulate B cells from untreated patients with RRMS and healthy donors. Moreover, we took advantage of the unique ability of the monoclonal antibody MK16 to recognize the immunodominant peptide MBP85-99 presented on HLA-DR15, and used it as a probe to directly study B-cell presentation of self-antigenic peptide. The proportions of B cells producing TNF-α or IL-6 after stimulation with MBP were higher in RRMS patients than in healthy donors, indicating a pro-inflammatory profile for self-reactive patient B cells. In contrast, polyclonal stimulation with PMA + ionomycin and MBP revealed no difference in cytokine profile between B cells from RRMS patients and healthy donors. Expanded disability status scale (EDSS) as well as multiple sclerosis severity score (MSSS) correlated with reduced ability of B cells to produce IL-10 after stimulation with MBP, indicative of diminished B-cell immune regulatory function in patients with the most severe disease. Moreover, EDSS correlated positively with the frequencies of TNF-α, IL-6 and IL-10 producing B cells after polyclonal stimulation. Patient-derived, IL-10-producing B cells presented MBP85-99 poorly, as did IL-6-producing B cells, particulary in the healthy donor group. B cells from MS patients thus present antigen to T cells in a pro-inflammatory context. These findings contribute to understanding the therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.

Characterization of Regulatory B Cells in Graves' Disease and Hashimoto's Thyroiditis.

A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups and correlated with free T3 levels in GD patients. IL-10+ B cells from both patient groups displayed CD25 or TIM-1 more frequently than did those from healthy donors. B-cell expression of two surface marker combinations previously associated with regulatory B-cell functions, CD24hiCD38hi and CD27+CD43+, did not differ between patients and healthy donors. In conclusion, our findings indicate that autoimmune thyroiditis is not associated with reduced frequency of IL-10+ B cells. These results do not rule out regulatory B-cell dysfunction, however. The observed phenotypic differences between IL-10+ B cells from patients and healthy donors are discussed.

Uptake and presentation of myelin basic protein by normal human B cells.

B cells may play both pathogenic and protective roles in T-cell mediated autoimmune diseases such as multiple sclerosis (MS). These functions relate to the ability of B cells to bind and present antigens. Under serum-free conditions we observed that 3-4% of circulating B cells from healthy donors were capable of binding the MS-associated self-antigen myelin basic protein (MBP) and of presenting the immunodominant peptide MBP85-99, as determined by staining with the mAb MK16 recognising the peptide presented by HLA-DR15-positive cells. In the presence of serum, however, the majority of B cells bound MBP in a complement-dependent manner, and almost half of the B cells became engaged in presentation of MBP85-99. Even though complement receptor 1 (CR1, CD35) and CR2 (CD21) both contributed to binding of MBP to B cells, only CR2 was important for the subsequent presentation of MBP85-99. A high proportion of MBP85-99 presenting B cells expressed CD27, and showed increased expression of CD86 compared to non-presenting B cells. MBP-pulsed B cells induced a low frequency of IL-10-producing CD4+ T cells in 3 out of 6 donors, indicating an immunoregulatory role of B cells presenting MBP-derived peptides. The mechanisms described here refute the general assumption that B-cell presentation of self-antigens requires uptake via specific B-cell receptors, and may be important for maintenance of tolerance as well as for driving T-cell responses in autoimmune diseases.

Depletion of T lymphocytes is correlated with response to temozolomide in melanoma patients.

Therapeutic strategies to deplete lymphocytes, especially regulatory T cells, in cancer patients have been proposed to increase the benefits of (immuno)chemotherapy. In this study, we explored the influence of temozolomide (TMZ) on different T-cell populations and addressed if the depletion of CD4+ T cells would be associated to the clinical benefits of TMZ. Patients were treated with TMZ (150 mg/m2 daily, every two weeks on a four-week schedule) until disease progression. Changes in T-lymphocyte subsets were characterized by flow cytometry. All patients enrolled in this study had histologically verified unresectable stage IV melanoma. Objective responses were induced in 12.5% of the patients, while 42.5% of them obtained short-term disease stabilization. The median progression-free survival (PFS) of this patient cohort was 8.7 mo. Lymphopenia (< 0.7 × 109 cells/L, grade 2) developed in 71% of the patients after 3 treatment cycles (~100 d). The development of grade 2 lymphopenia after the 3rd cycle of therapy positively correlated with clinical outcome (p = 0.01), and was linked, though non-significantly, to prolonged median PFS (303 vs. 200 d). In addition, significant changes in CD8+ T-cell subgroups were observed, notably a shift from naïve T cells toward more differentiated memory T cells. Finally, we demonstrated that specific CD8+ T-cell responses against selected tumor associated antigens (TAAs) were enhanced by the administration of TMZ (p = 0.04), while virus-specific T-cell responses were stable. Thus, immunological monitoring in the course of TMZ treatment might become an important tool for clinical guidance in the future.

Increase of circulating CD4+CD25highFoxp3+ regulatory T cells in patients with metastatic renal cell carcinoma during treatment with dendritic cell vaccination and low-dose interleukin-2.

Regulatory T cells (Treg) play an important role in the maintenance of immune tolerance and may be one of the obstacles of successful tumor immunotherapy. In this study, we analyzed the impact of administration of dendritic cell (DC) vaccination in combination with low-dose interleukin (IL)-2 in patients with metastatic renal cell carcinoma on the frequency of CD4+CD25highFoxp3+ Treg cells in peripheral blood. We found that the treatment increased the frequency of Treg cells more than 7-fold compared with pretreatment levels (P<0.0001). The frequency of Treg cells decreased when patients had been off IL-2 treatment for only 8 days, but remained higher than pretreatment levels. A functional assay showed that isolated Treg cells were capable of inhibiting proliferation of responder cells. Also, in vitro studies showed that coculture of mature DCs, autologous T cells and IL-2 leads to an increase in the number of Treg cells whereas IL-21 does not stimulate the induction of Treg cells. These findings demonstrate that even low doses of IL-2 in combination with DC vaccination are able to expand CD4+CD25+Foxp3+ Treg cells in vivo in metastatic renal cell carcinoma patients. Further, the results indicate that the IL-2-induced effect on Treg cells is reversible and declines shortly after termination of IL-2 treatment. Our data suggest that approaches combining DC-mediated immunotherapy and depletion of Treg cells may be necessary to enhance the ability of vaccination therapy to elicit effective antitumor responses in cancer patients. Also, adjuvant IL-21 administration may lead to immune enhancement without simultaneous induction of Treg cells.

Identification of heme oxygenase-1-specific regulatory CD8+ T cells in cancer patients.

Treg deficiencies are associated with autoimmunity. Conversely, CD4+ and CD8+ Tregs accumulate in the tumor microenvironment and are associated with prevention of antitumor immunity and anticancer immunotherapy. Recently, CD4+ Tregs have been much studied, but little is known about CD8+ Tregs and the antigens they recognize. Here, we describe what we believe to be the first natural target for CD8+ Tregs. Naturally occurring HLA-A2-restricted CD8+ T cells specific for the antiinflammatory molecule heme oxygenase-1 (HO-1) were able to suppress cellular immune responses with outstanding efficacy. HO-1-specific CD8+ T cells were detected ex vivo and in situ among T cells from cancer patients. HO-1-specific T cells isolated from the peripheral blood of cancer patients inhibited cytokine release, proliferation, and cytotoxicity of other immune cells. Notably, the inhibitory effect of HO-1-specific T cells was far more pronounced than that of conventional CD4+CD25+CD127- Tregs. The inhibitory activity of HO-1-specific T cells seemed at least partly to be mediated by soluble factors. Our data link the cellular stress response to the regulation of adaptive immunity, expand the role of HO-1 in T cell-mediated immunoregulation, and establish a role for peptide-specific CD8+ T cells in regulating cellular immune responses. Identification of potent antigen-specific CD8+ Tregs may open new avenues for therapeutic interventions in both autoimmune diseases and cancer.

Low expression of insulin in the thymus of non-obese diabetic mice.

Insulin is a predominant autoantigen in IDDM in man and the NOD mouse. Failure of negative selection of diabetogenic T cells in thymus may be an important pre-disposing cause of the disease. To obtain insight into negative selection against such T-cell clones the thymic expression of insulin was studied in NOD and Balb/c mice by quantitative competitive RT-PCR. We detected RNA for insulin in the thymus of 3-week-old Balb/c mice as well as in NOD mice. However, the NOD mice expressed only half as many insulin transcripts as the Balb/c mice. Also, insulin protein was detected in the thymic medulla of both Balb/c and NOD mice. Furthermore, thymic RNA preparations were investigated for the presence of insulin transcription factors. None of the known pancreatic transcription factors for insulin; Pdx-1, Pax6 or Nkx6.1 were detectable in the thymus of Balb/c mice. These results support the idea that low insulin expression in the thymus may be a predisposing cause for development of diabetes in NOD mice analogous with what has been found in humans with the disease-disposing IDDM2 allele. Furthermore, our results suggest that insulin expression in the thymus may be regulated by different principles from those in the pancreas.