Charlotte Christie Petersen

TLR3 Ligand Polyinosinic:Polycytidylic Acid Induces IL-17A and IL-21 Synthesis in Human Th Cells

TLR3 and TLR9 recognize the pathogen-associated microbial patterns dsRNA and unmethylated DNA, respectively. The recent discovery that these receptors also recognize endogenous ligands from necrotic material has drawn increased attention to their involvement in autoimmunity. Th cell cytokines IL-17A and IL-21 have been assigned with pivotal roles in the regulation of such autoimmune diseases. IL-17A is the hallmark cytokine of the recently discovered proinflammatory Th cell subset TH17. By contrast, the expression of IL-21 does not seem to be limited to a single distinct Th cell subset. We investigated the expression of IL-17A and IL-21 in human CD4+ T cells in response to stimulation with the TLR3 ligand polyinosinic:polycytidylic acid (poly(I:C)) and the TLR9 ligand CpG. We discovered that poly(I:C) induced synthesis of both IL-17A and IL-21. Moreover, we found that poly(I:C) was able to drive the differentiation of naive Th cells into an IL-21 but not into an IL-17A-producing phenotype and did this without affecting the levels of transcription factors T-bet, GATA-3, or retinoic acid receptor-related orphan receptor C. Finally, we found that the IL-21-producing cells that were differentiated in response to poly(I:C) expressed the chemokine receptor CXCR3, which is important in the recruitment of T cells into inflamed joints in rheumatoid arthritis. This is the first report to show that the TLR3 ligand poly(I:C) can directly induce the synthesis of IL-17A and IL-21 and drive differentiation of human naive CD4+ T cells.

T cell homing to tumors detected by 3D-coordinated positron emission tomography and magnetic resonance imaging.

A general hindrance to progress in adoptive cellular therapy is the lack of detailed knowledge of the fate of transferred cells in the body of the recipient. In this study, we present a novel technique for tracking of 124I-labeled cells in situ, which combines the high spatial resolution of magnetic resonance imaging with the high sensitivity and spatial accuracy of positron emission tomography. We have used this technique, together with determination of tissue radioactivity, flow cytometry, and microscopy, to characterize and quantitate the specific accumulation of transferred CD8+ T cells in tumor tissue in a mouse model. Transgenic CD8+ T cells, specific for the ovalbumin peptide SIINFEKL, were adoptively transferred to recipients carrying a subcutaneous tumor of the ovalbumin-expressing malignant melanoma cell line B16-OVA. The number of SIINFEKL-specific CD8+ cells in the tumor tissue was determined by flow cytometry each day for 8 consecutive days after adoptive transfer. From low levels 1 day after injection, their number gradually increased until day 5 when an average of 3.3×106 SIINFEKL-specific cells per gram tumor tissue was found. By applying the combined positron emission tomography/magnetic resonance imaging technique we were able to determine the position of the transferred, 124I-labeled SIINFEKL-specific T cells in 3 dimensions in recipient mice, and could demonstrate a highly significant accumulation of the 124I label in and around the subcutaneous B16-OVA tumors compared with normal tissue. Accumulation of 124I was significantly higher in B16-OVA than in B16 tumors not expressing the OVA antigen.

Accumulation in tumor tissue of adoptively transferred T cells: A comparison between intravenous and intraperitoneal injection

Accumulation of T cells at the tumor is essential in cancer immunotherapy based on adoptive transfer of tumor-specific T cells. To gain further insight into the accumulation process and to evaluate the effect of using different routes of cell transfer, we investigated the accumulation of ovalbumin-specific CD8+ T cells (OT-I) injected either intravenously (IV) or intraperitoneally (IP) into mice carrying a subcutaneous tumor of the ovalbumin-expressing melanoma cell line B16-OVA. Maximal accumulation of the adoptively transferred cells in tumor tissue was observed 5 days after injection, irrespective of the injection route. The route of injection affected neither the total number of adoptively transferred cells found in tumor tissue nor the kinetics of this accumulation. In the spleen, however, the accumulation of adoptively transferred cells was clearly dependent on the injection route. IP injections resulted in a large number of adoptively transferred cells in the spleen on all days analyzed. In comparison, IV injection resulted in significantly fewer adoptively transferred cells in the spleen, and this number decreased over time. The route of injection affected neither the activation status of the adoptively transferred T cells that accumulated at the tumor site, nor the ability of these cells to control tumor growth. Two cell populations, SIINFEKL-tetramerLow(TetLow)CD69+CD25+ and TetHighCD69−CD25−, were present in tumor samples, whereas only TetHighCD69−CD25− cells accumulated in the spleen. In tumors, IV injection resulted in a higher fraction of adoptively transferred cells with an activated phenotype (TetLowCD69+CD25+) compared with IP injection.

The B1-cell subpopulation is diminished in patients with relapsing–remitting multiple sclerosis

B cell subsets in newly diagnosed untreated, relapsing-remitting multiple sclerosis (MS) patients were examined. The fraction of CD20(+) B cells was significantly increased in MS. Among subsets of B cells, MS patients had increased frequency of naïve cells, but reduced frequency of memory and B1 cells. The frequencies of B1 cells were inversely correlated with the time since last attack. B1 cells resembled the phenotype of either lymphocytes (CD11b(-) B1 cells) or monocytes (CD11b(+) B1 cells) and a small fraction of cells was CD3(+)CD20(+) by confocal microscopy.

Common variable immunodeficiency unmasked by treatment of immune thrombocytopenic purpura with Rituximab

BackgroundHypogammaglobulinemia may be part of several different immunological or malignant conditions, and its origin is not always obvious. Furthermore, although autoimmune cytopenias are known to be associated with common variable immunodeficiency (CVID) and even may precede signs of immunodeficiency, this is not always recognized. Despite novel insight into the molecular immunology of common variable immunodeficiency, several areas of uncertainty remain. In addition, the full spectrum of immunological effects of the B cell depleting anti-CD20 antibody Rituximab has not been fully explored. To our knowledge this is the first report of development of CVID in a patient with normal immunoglobulin prior to Rituximab treatment.Case presentationHere we describe the highly unusual clinical presentation of a 34-year old Caucasian male with treatment refractory immune thrombocytopenic purpura and persistent lymphadenopathy, who was splenectomized and received multiple courses of high-dose corticosteroid before treatment with Rituximab resulted in a sustained response. However, in the setting of severe pneumococcal meningitis, hypogammaglobulinemia was diagnosed. An extensive immunological investigation was performed in order to characterize his immune status, and to distinguish between a primary immunodeficiency and a side effect of Rituximab treatment. We provide an extensive presentation and discussion of the literature on the basic immunology of CVID, the mechanism of action of Rituximab, and the immunopathogenesis of hypogammaglobulinemia observed in this patient.ConclusionsWe suggest that CVID should be ruled out in any patient with immune cytopenias in order to avoid diagnostic delay. Likewise, we stress the importance of monitoring immunoglobulin levels before, during, and after Rituximab therapy to identify patients with hypogammaglobulinemia to ensure initiation of immunoglobulin replacement therapy in order to avoid life-threatening invasive bacterial infections. Recent reports indicate that Rituximab is not contra-indicated for the treatment of CVID-associated thrombocytopenia, however concomitant immunoglobulin substitution therapy is of fundamental importance to minimize the risk of infections. Therefore, lessons can be learned from this case report by clinicians caring for patients with immunodeficiencies, haematological diseases or other autoimmune disorders, particularly, when Rituximab treatment may be considered.

Short-term exposure to human cytomegalovirus-infected fibroblasts induces a proportional increase of active CD94/NKG2A+ natural killer cells

Natural killer (NK) cells are essential components of the immune response against human cytomegalovirus (HCMV). As NK cells are part of the innate immune system providing an immediate defense against pathogens, short-term exposure to HCMV-infected cells may induce changes in the phenotype and function of these cells. To identify immediate reactions of NK cells to HCMV, we co-cultured peripheral blood mononuclear cells with HCMV-infected fibroblasts for 24 and 72 hours. A distinct, HCMV-mediated, proportional enlargement of a subset of NK cells expressing CD94/NKG2A was sustained throughout the period of incubation. As preceding studies have shown that HCMV can cause an increase in CD94/NKG2C(+) NK cells, our results were surprising. The NK cells showed intense upregulation of the early activation marker CD69 in response to HCMV. The CD94/NKG2A(+) NK cells demonstrated the highest expression of CD69. Studies of HCMV-induced interferon-gamma expression after 24 hours of co-culture showed that this cytokine was almost exclusively produced by the CD94/NKG2A(+) subset of NK cells. In summary, our data demonstrate that HCMV induces an immediate proportional enlargement of a functionally active CD94/NKG2A expressing subset of NK cells.

Interleukin-21 restrains tumor growth and induces a substantial increase in the number of circulating tumor-specific T cells in a murine model of malignant melanoma

New strategies of immunotherapy are currently being evaluated, and the combination of chemo- and immunotherapy has shown promising results. The cytokine interleukin-21 (IL-21) is known to enhance immune function, and in this study we have investigated its ability to boost the efficacy of chemoimmunotherapy-cyclophosphamide and adoptive cell transfer (ACT)-in the B16-OVA/OT-I murine model of malignant melanoma. Subcutaneous B16-OVA tumors were established in C57BL/6J mice 8 days before adoptive transfer of tumor-specific OT-I T cells. In addition to cyclophosphamide and ACT, one group of mice received daily injections of murine IL-21 (mIL-21). Mice treated with mIL-21 had more tumor-specific T cells in the circulation 4 and 7 days following ACT (P=0.004 and P=0.002, respectively). Importantly, mIL-21 and ACT controlled tumor growth instantly and more effectively than ACT alone (P=0.001, day 4)-an effect that persisted up to 5 days after the last mIL-21 injection. We conclude that mIL-21 enhances chemoimmunotherapy: it amplifies the number of tumor-specific T cells in the circulation and also stunts early tumor growth.

Human herpesvirus 6B induces phenotypic maturation without IL-10 and IL-12p70 production in dendritic cells.

Human herpesvirus 6B (HHV‐6B) is the causative agent of the common childhood febrile illness, exanthema subitum. The virus is predominantly regarded as a T‐cell tropic virus, although in reality it has the ability to infect a wide variety of cell types including monocytes, macrophages and dendritic cells (DC). Although DC are important immune regulators, the modulating effects of HHV‐6B on DC are controversial. Here, we examine the phenotypic and functional consequences of HHV‐6B infection of DC. The addition of HHV‐6B to immature DC led to expression of the nuclear viral p41 protein and cell surface expression of the viral glycoprotein gp60/110 consistent with HHV‐6B infection. Nevertheless, HHV‐6B did not induce noticeable cytopathogenic effects or cell death in infected DC. Importantly, HHV‐6B infection induced a partial phenotypic maturation of immature DC as demonstrated by a substantial increase in the expression of HLA‐DR, CD86 and CD40, whereas only a minor increase in CD80 and CD83 was observed. This phenotypic maturation was, however, not followed by functional maturation, because HHV‐6B infection did not induce IL‐10 and IL‐12p70 production in immature DC. However, infected DC were still able to react to bacteria‐derived stimuli such as lipopolysaccaharide by an even more pronounced production of IL‐10 and IL‐12p70 when compared to that of uninfected DC.

Increased expression of CD69 on T cells as an early immune marker for human cytomegalovirus reactivation in chronic lymphocytic leukemia patients.

Reactivation of human cytomegalovirus (HCMV) remains a serious problem in immunosuppressed individuals. To investigate whether a change in the immune status can be used as an earlier marker for HCMV reactivation than the traditional PCR analysis, eight chronic lymphocytic leukemia (CLL) patients at risk for reactivation due to commencement of alemtuzumab (anti-CD52) treatment were longitudinally followed. Five series of consecutive weekly blood samples were immunophenotyped by flow cytometry to cover both the innate and adaptive immune responses. Concurrently, patients were monitored by PCR for HCMV reactivation. We found a minor upregulation of the early activation marker CD69 on NK cells immediately before HCMV was detected in circulation by PCR. Interestingly, for the specific immune response, CD69 was highly upregulated on CD3(+) T cells, especially for the CD8(+) subset, in the two patients experiencing an HCMV reactivation between 6 and 20 d before HCMV viremia was measured by PCR. Moreover, a CD4(+):CD8(+) ratio lower than 0.6 may indicate a trend toward an increased risk for viral reactivation. In conclusion, an increase in CD69 expression is a promising candidate as an early predictor of HCMV reactivation.

Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission

The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34+ stem cell and progenitor cell subpopulations were isolated by fluorescence‐activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph+) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4‐log reduction in BCR‐ABL1 expression (MR4), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA‐based qPCR in detecting residual Ph+ stem cells (P = 0·005), and detected Ph+ stem‐ and progenitor cells in 9/10 patients at frequencies of 2–14%. Moreover, while all qPCR+ samples also were iFISH+, 9/33 samples were qPCR‐/iFISH+, including all positive samples from MR4 patients. Our findings show that residual Ph+ cells are low BCR‐ABL1 producers, and that DNA‐based methods are required to assess the content of persisting Ph+ stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR4, and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.