Pneumococcal polysaccharide abrogates conjugate-induced germinal center reaction and depletes antibody secreting cell pool, causing hyporesponsiveness.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.Plain pneumococcal polysaccharide (PPS) booster administered during second year of life has been shown to cause hyporesponsiveness. We assessed the effects of PPS booster on splenic memory B cell responses and persistence of PPS-specific long-lived plasma cells in the bone marrow (BM).Neonatal mice were primed subcutanously (s.c.) or intranasally (i.n.) with pneumococcal conjugate (Pnc1-TT) and the adjuvant LT-K63, and boosted with PPS+LT-K63 or saline 1, 2 or 3 times with 16 day intervals. Seven days after each booster, spleens were removed, germinal centers (GC), IgM(+), IgG(+) follicles and PPS-specific antibody secreting cells (AbSC) in spleen and BM enumerated.PPS booster s.c., but not i.n., compromised the Pnc1-TT-induced PPS-specific Abs by abrogating the Pnc1-TT-induced GC reaction and depleting PPS-specific AbSCs in spleen and limiting their homing to the BM. There was no difference in the frequency of PPS-specific AbSCs in spleen and BM between mice that received 1, 2 or 3 PPS boosters s.c.. Repeated PPS+LT-K63 booster i.n. reduced the frequency of PPS-specific IgG(+) AbSCs in BM.PPS booster-induced hyporesponsiveness is caused by abrogation of conjugate-induced GC reaction and depletion of PPS-specific IgG(+) AbSCs resulting in no homing of new PPS-specific long-lived plasma cells to the BM or survival. These results should be taken into account in design of vaccination schedules where polysaccharides are being considered.Icelandic Research Fund for Graduate Students/ 50940005, Icelandic Research Fund/40438021-23, Eimskip University Fund, University of Iceland Research Fund, Landspitali University Hospital Research Fun

Harnessing Dendritic Cell Reprogramming to Elucidate Mechanisms of Tumor Immunity

The presence of conventional dendritic cells type 1 (cDC1) in the tumor correlates with positive treatment outcome. The ability to cross-present neoantigens and prime protective CD8+ T-cell responses, makes cDC1s central for tumor immunity. However, in tumors cDC1 are rare and often functionally impaired. Our group reported that overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) converts mouse and human fibroblasts into cross-presenting cDC1-like cells. We employed the minimal gene regulatory network of highly immunogenic cDC1 and restored the immunogenicity of low immunogenic lung cancer and melanoma cell lines by reprogramming into professional tumor antigen presenting cells (tumor-APCs). Here, we report that upon transduction with PIB, 23 solid syngeneic cancer lines initiate reprogramming into cDC1-like cells expressing CD45 and MHC-II at efficiencies ranging from 0.5-57.7%. Functionally, PIB overexpression endows tumor cells with the capacity to cross-present exogenous antigen and prime naïve CD8+ T-cells. Adoptive transfer of ovalbumin cross-presenting B16 tumor-APCs into established ovalbumin expressing B16 tumors (B16-OVA) elicits tumor growth control and extends animal survival. Treated animals show a systemic antigen-specific T cell response against ovalbumin and endogenous tumor-associated antigen MuLV p15E. Intratumoral injection of reprogrammed B2905 and LLC into tumors shows differential response, correlating with their cross-presentation capacity. This approach combines cDC1 antigen cross-presentation abilities with the generation of tumor antigens. The induction of a cDC1 identity in tumor cells sets in motion T cell responses in vitro and in vivo. In the future of this project, dendritic cell reprogramming will be object in a 2-cell CRISPR/Cas9 screen using induced cDC1-like tumor cells and reporter T-cells to explore mechanistically cross-presentation regulators. The generation of cross-presenting tumor-APCs will be also used to map and characterize presented and cross-presented neoantigens. Finally, dendritic cell reprogramming of tumor cells will be explored in vivo by replenishing cDC1 within the tumor microenvironment through in vivo reprogramming. Ultimately, this project will provide insight into mechanisms of cross-presentation and pave the way for the development of novel cDC1-centric therapies

Immunogenicity of rat-neu(+) mouse mammary tumours determines the T cell-dependent therapeutic efficacy of anti-neu monoclonal antibody treatment

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2(+) breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu(+) tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.Functional Genomics of Systemic Disorder

A Restricted Role for FcγR in the Regulation of Adaptive Immunity.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity

Mechanisms of melanoma-targeting antibody therapy in mice

The aim of the research described in this thesis was to gain a better understanding of the role of the different immune cells and the different FcR on their cell surface, in antibody therapy and to investigate whether the effectiveness of the tumor-killing mechanisms, activated by the antibodies, can be improved. First, we investigated the role of FcR in various immune responses using a genetically modified mouse, in which all FcR were missing. After defining that role, we studied antibody therapy in a mouse model for melanoma in two different ways: on the one hand, after vaccination using a viral vector that expressed a melanoma antigen, on the other hand, by injecting a melanoma-specific antibody in combination with other substances that activate the immune system. The research described in this thesis was performed at the Department of Human Genetics, the Department of Medical Oncology and the Department of Immunohematology and Bloodtransfusion of the Leiden University Medical Center, Leiden, The Netherlands and was supported by funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013 under Grant Agreement 317445LUMC / Geneeskund

Subcutaneous administration of PPS-1 booster depletes Pnc1-TT-induced PPS-1-specific AbSC pool in the spleen.

<p>PPS-1 (upper panels) and TT (lower panels) specific IgG⁺ AbSC measured by ELISPOT in spleen (A and B) and bone marrow (C and D), shown as number of spots (mean±SD) per 10⁶ spleen cells and PPS-1- and TT-specific IgG levels (mean EU/ml±SD) in serum measured by ELISA (E and F). Results are shown for mice boosted by either i.n. or s.c. route with saline (open columns), PPS-1+LT-K63 (black columns), Pnc1-TT+LT-K63 (grey columns) and unvaccinated controls (stribe columns), as indicated. Statistical difference between vaccinated groups is indicated in the graphs. All vaccinated mice had significantly higher frequency of PPS-1 -specific IgG⁺ AbSCs in spleen and BM and also higher serum IgG anti-PPS-1 levels compared to unvaccinated controls (B–F), except mice that received PPS-1 booster s.c. which had comparable frequency of PPS-1-specific IgG⁺ AbSCs in spleen (A). The results shown in A–F are from one of two independent experiments showing comparable results (8 mice per group in each experiment).</p

Plain PPS-1 booster abrogates the Pnc1-TT-induced GC reaction in mice primed as neonates.

<p>(A–C) Mean number of follicles and (D) ratio of GC/follicle per section in consecutive sections from spleen of mice that received booster by either s.c. (left columns) or i.n. (right columns) route with saline (open columns), PPS-1+LT-K63 (black columns), Pnc1-TT+LT-K63 (grey columns) and unvaccinated controls (stribe columns), as indicated. Spleens were removed 7 days after booster in mice primed as neonates with Pnc1-TT+LT-K63 by the same route as the booster. Half of the spleen was snap frozen, serial cryosection prepared, cutting into 7 µm sections at four levels, starting 700 µm into the tissue and the levels separated by 210 µm. Section from all 4 levels were stained with (A) anti-IgM, (B) anti-IgG, (C) PNA, and results (mean±SD) shown for each group. (D) Mean ratio of GC/follicle was calculated for every spleen at all 4 levels for individual mice and results (mean±SD) shown for each group. Statistical difference between vaccinated groups is stated in the graphs. Results in A–D are from one representative of two independent experiments (8 mice per group) showing comparable results.</p

PPS-1 booster abrogation of Pnc1-TT-induced GCs reduces levels, avidity and protective efficacy of PPS-1-specific IgG.

<p>PPS-1-specific IgG levels (mean EU/ml±SD) in serum (A) and their avidity index (mean AI±SD) (B) measured by ELISA −2 days before and 7, 23 and 39 days after s.c. (left panels) or i.n. (right panels) booster with saline (open squares), PPS-1+LT-K63 (filled squares) in mice primed with Pnc1-TT as neonates or unvaccinated controls (open circles). Six weeks after the booster the mice were challenged with live <i>S. pneumoniae</i> serotype 1 to assess protection against bactermia and lung lung infection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072588#pone.0072588-Jakobsen1" target="_blank">[26]</a>. Pneumococcal colony forming units (CFU/ml) in blood (C, left graph) and lungs (C, right graph) 24 h after challenge shown for each mouse (N = 8/group), the median for each group is indicated by a line. Statistical difference in bacteremia or lung infection between vaccinated groups is indicated in the graphs. The results shown are from one of two independent experiments (eight mice/group for each time point) showing comparable results.</p

The adjuvant LT-K63 can restore delayed maturation of follicular dendritic cells and poor persistence of both protein- and polysaccharide-specific antibody-secreting cells in neonatal mice.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT-induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2(+) staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1(+) macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2(+) FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG(+) AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.Icelandic Research Fund Landspitali University Hospital Research Fund University of Iceland Research Fund European Commission Icelandic Research Fund for Graduate Students Eimskip Fund of the University of Icelan

A single PPS-1 booster s.c. is sufficient to completely deplete PPS-1-specific-memory established by neonatal Pnc1-TT-priming.

<p>PPS-1-specific IgG⁺ AbSCs, shown as number of spots (mean±SD) per 10⁶ cells, in spleen (A) and bone marrow (B) measured by ELISPOT, and PPS-1-specific IgG Abs (mean EU/ml±SD) in serum (C) measured by ELISA, at day 7, 23 and 39 after mice receiving s.c. booster with saline, PPS-1+LT-K63 or unvaccinated controls. Statistical difference between test groups and unvaccinated controls are indicated as; * P<0.05; ** P≤0.001, and the difference between vaccinated groups is stated in the graphs. The results are shown for one of two independent experiments (eight mice/group for each time point), showing comparable results.</p