The reaction of the immune system of fish to vaccination

The studies presented in this thesis deal with the effect of bacterial antigens of Yersinia ruckeri and Aeromonashydrophila on the immune system of carp. The antigens were administered by injection or by bath treatment. The effect on the immune system was studied by measuring the numbers of antibody forming cells (AFCs), the level of serum antibody and the processing of antigen in the lymphoid organs. The antigen dose and the formation of immunological memory were taken into account. Moreover, in view of oral vaccination, a study was carried out on the uptake and transport of macromolecules through the intestinal epithelial cells.Y. ruckeri O-antigen was very immunogenic in carp. Both i.m. and i.p. injection of antigen and direct immersion in antigen solution evoked distinct levels of AFCs and serum antibody (Appendix paper I). The height of the response was dependent on entry route; e.g. it was clearly lower after direct immersion. Contrary to the numbers of AFCs, which showed a fast decrease after a peak at day 10, serum antibody persisted for a long time, especially at the highest antigen dose. It was suggested that a long-term presence of antigen in the lymphoid organs, as observed by immunofluorescence, might account for this feature.Secondary antibody responses in carp injected or bathed in Y.ruckeri O-antigen gave indications for the formation of immunological memory on the first contact with the antigen for both priming routes. Highest secondary responses were obtained in animals in which antibody levels resulting from the priming had decreased to background levels (Appendix paper II).Upon i.m. injection of two A.hydrophila bacterins, formalin killed cells (F-A h ) and heat killed and disrupted cells (H-A h ), considerable serum antibody titers were found in carp. H-A h induced higher levels of antibody than F-A h , which in both cases persisted for more than 8 months (Appendix paper III). Whereas antigen dose clearly affected the height of the response, the peak day depended on the type of bacterin (day 14 and day 20 for all doses of H-A h and F-A h respectively). The effect of mixing the antigen with adjuvant found expression in a second increase of AFCs after a "normal" peak in the primary response, and a prolonged increase of serum antibody levels (Appendix paper VIII).Antisera raised against the two A.hydrophila b acterins showed different agglutinating properties (Appendix paper III). Antibody induced by F-A h was mainly directed to lipopolysaccharide (LPS), whereas with H-A h this was not the case. It was concluded that LPS is an important antigen of A. hydrophila. However, there is at least one other immunogenic component.Fractionated immune (anti F-A h ) and non-immune control sera were tested for immunoglobulin (Ig) by an Elisa and for agglutinating properties. It was shown that Ig was restricted to the first high molecular weight protein peak. In addition to the antibody in this peak also a non-Ig agglutinin was present in lower molecular weight fractions, probably a natural agglutinin.Memory formation was monitored by studying secondary responses upon a primary i.m. injection, carp developed memory to A.hydrophila bacterin (Appendix paper VI). The height of the secondary responses was directly correlated with the priming dose. Moreover, the dose of the booster injection determined the outcome of the formed memory; corresponding priming and boosting doses expressed the highest memory levels. The development of maximum memory took some months, and its presence was still demonstrable at 12 months. The low dose priming induced only a weak memory, which was limited in time.A single bath in A.hydrophila bacterin was not followed by the appearance of serum antibody. However, it did induce formation of memory, for a second bath resulted in clear secondary responses. This memory was maximum at 3 months after priming and was limited in time, for it was gone at 12 months. When a booster was given by i.m. injection instead of immersion, the induced memory was not expressed as a clear secondary response. This feature suggests the importance of local processes during the immune response after bath vaccination (Appendix paper V).In Appendix paper VI a brief morphological description is given of the spleen, head kidney and trunk kidney. An antigen localization study was carried out after i.m. injection of A.hydrophila bacterin, by means of immunofluorescence. The antigen first appeared in the splenic ellipsoids and solitary phagocytic cells of the head and trunk kidney. Later on it gradually concentrated in or near melanocentres (MMCs) in all organs studied. In the later phases antigen was only located in and around MMCs, both intracellularly and bound to cell membranes, and it remained so for a full year. No antigen could be traced in the lymphoid organs following bath immunization. The question arose if the observed processes were of immunological interest, and which were just a non-specific reaction. Therefore the fate of injected inert material (carbon) was studied for comparison (Appendix paper VII). The processing of carbon was comparable to that described for A.hydrophila antigen. However, at two points there was a difference: 1) in the spleen carbon was transported to MMCs by cells leaving the ellipsoids, whereas this was not clear for A.hydrophila antigen, which appeared near and in MMCs bound to the surface of cells; 2) carbon was only seen intracellularly. Based on these observations it was assumed that the membrane bound antigen in the MMCs had immunological relevance. To test this assumption simultaneous histological and immunological tests were carried out after injection of A. hydrophila antigen (Appendix paper VIII). In those tests it was found that extracellular bound antigen in MMCs was not observed until circulating antibody was present, which is suggestive for the antigen to be complexed by antibody. A further support for this assumption was given by the simultaneous presence of tissue bound Ig and A.hydrophila antigen in MMCs.The possible immunological importance of MMCs is discussed in Appendix papers VI-VIII. There are several indications for MMCs to be early phylogenetic analogues of the mammalian germinal centres.In Appendix paper IX a study is presented on uptake and transport of intact macromolecules in the intestinal epithelium of carp.This study was started as oral administration is an interesting vaccination method. Electronmicroscopical observations showed that two protein tracer-molecules, horse radish peroxidase (HRP) and ferritin were absorbed more extensively in the second than in the first gut segment. Moreover, HRP and ferritin were processed by the enterocytes in a different way. HRP seemed to be taken up by membrane binding and was subsequently transported to the intercellular space, where it contacted the abundant intra-epithelial leucocytes. There was no notable intracellular digestion of HRP. However, ferritin was taken up by pinocytosis and ended up in lysosome-like bodies or supranuclear vacuoles (2nd segment), where it was digested slowly. Although no ferritin was found in the intercellular space, large macrophages, with phagosomes containing ferritin, were present between the epithelial cells. Also some smaller macrophages containing ferritin, have been observed in the lamina propria and even in the spleen. These data were discussed in terms of both selective and non-selective absorption of macromolecules and the possible immunological implications. It was concluded that orally administered antigens reach intra-epithelial leucocytes, and might induce a (local) immune response. As most antigens are transmitted in the second segment, this part of the gut probably has an important immunological function.FINAL CONCLUSIONS- The humoral immune response in carp can be evoked by soluble and particulate bacterial antigens from Yersinia ruckeri and Aeromonas hydrophila.- The height of the humoral response and the antigen dose are directly correlated.- The height of the humoral response is dependent of the route of antigen administration. Injection gives higher serum antibody levels than bath vaccination.- The peak day of the response is not antigen dose dependent, but is influenced by the bacterin type.- Circulating antibody in these animals can be very persistent (e.g. 1 year). This phenomenon is due to continuous stimulation by antigen in or on macrophages (especially melano-macrophages).- Local processes in the skin and gills play a regulatory role in the response after bath vaccination.- Memory develops after both antigen injection and bathing in antigen solution.- The development of memory in fish takes much more time than the antibody response itself. Maximum levels are usually not reached before 3 months after the initial contact with the antigen.- The height of the secondary responses can be used as an indirect method for the quantification of memory. The data obtained can be used for calculating a memory factor.- The height of memory levels achieved and the priming antigen dose are directly correlated.- The expression of memory is affected by several factors, such as residual "priming" antibody at the moment of boosting, the ratio between the first and second antigen dose, and whether or not the priming and boosting routes correspond.- In the early phase of the response mononuclear phagocytes in the kidney and spleen are responsible for antigen handling and stimulation of immuno-competent cells. In the later phase melano-macrophage centres (MMCs) become more important for the stimulation of the response and for memory induction.- MMCs probably are the early phylogenetic analogous of mammalian germinal centres.- Epithelial cells of the second segment of the intestine are important for the initial antigen uptake. Certain antigens are not digested in the cells, but released in the intercellular space (circulation) and taken up by intestinal macrophages. It is unclear if the intestinal lymphocytes react upon these antigens.- Successful application of bacterial vaccines to fish is possible. Injection methods can be used for small numbers of fish. Bath methods are possible for large scale application or young animals. Oral administration is not as effective as injection or immersion, but recent data provide good prospectives for future use.- The best results in terms of protection can be expected when the vaccination route (e.g. bath) corresponds with the route of pathogen entry (e.g. gills and skin).- In tests for the determination of vaccination efficacy the same challenge route as the vaccination route should be preferred.- It will be an advantage when fish, after a vaccination, can develop immunological memory during a period of 2-3 months at optimal temperatures in a relatively clean environment (e.g. specific pathogen free hatcheries).</p

T cell receptors for clinical therapy: in vitro assessment of toxicity risk

Adoptive therapy with T cell receptor (TCR)-engineered T cells has shown promising results in the treatment of patients with tumors, and the number of TCRs amenable for clinical testing is expanding rapidly. Notably, adoptive therapy with T cells is challenged by treatment-related side effects, which calls for cautious selection of target antigens and TCRs that goes beyond their mere ability to induce high T cell reactivity. Here, we propose a sequence of in vitro assays to improve selection of TCRs, and exemplify risk assessments of on-target as well as off-target toxicities using TCRs directed against Cancer Germline Antigens. The proposed panel of assays covers parameters considered key to safety, such as expression of target antigen in healthy tissues, determination of a TCR's recognition motif towards its cognate peptide, and TCR's cross-reactivity towards non-cognate peptides

Local but no systemic immunomodulation by intraperitoneal treatment of advanced ovarian cancer with autologous T lymphocytes re-targeted by a bi-specific monoclonal antibody

We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3+, 4-, 8+, 16-, 56-. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3+, 4-, 8+) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov- 1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation

Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv 18 (overexpressed by ovarian carcinoma cells) as part of a phase l/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (106−109) of bsAbtargeted T lymphocytes plus lowdose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 109 targeted T lymphocytes, 2 mg soluble bsAb, and lowdose IL-2. Using enzymelinked immunosorbent assays (ELISA), human antimouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (I) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human antimouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line lgrov-1 by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients

Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection

Recovery of cytomegalovirus (CMV)-specific T-cell-mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. The study used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV (NLV) peptide derived from the CMV protein pp65 to quantify A2-NLV-specific CD8+ T cells in partially T-cell-depleted grafts administered to 27 HLA-A*0201+ patients and to monitor recovery of these T cells during the first 12 months after SCT. None of the 9 CMV-seronegative patients became infected with CMV, whereas 14 of 18 CMV-seropositive patients developed CMV antigenemia after SCT. CMV-seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with ganciclovir (GCV) than those of grafts from CMV-seropositive donors (3 [1-6] versus 1 [0-3] courses, respectively; P =.009). The number of A2-NLV-specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = -0.61; P =.01). None of the 9 CMV-seronegative patients mounted a CMV-specific immune response as measured by monitoring A2-NLV-specific CD8+ T cells after SCT. Thirteen of 14 CMV-seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV-specific CD8+ T cells after SCT (P =.002). Thus, enumeration of HLA-restricted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells after SCT may constitute a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease

Inflammation and fatigue dimensions in advanced cancer patients and cancer survivors: An explorative study

BACKGROUND: Inflammation may underlie cancer-related fatigue; however, there are no studies that assess the relation between fatigue and cytokine

Cytokine and angiogenic factors associated with efficacy and toxicity of pazopanib in advanced soft-tissue sarcoma: An EORTC-STBSG study

Background: Pazopanib has activity in relapsed non-adipocytic soft-tissue sarcomas (STS). A series of serum cytokines and angiogenic factors (CAFs) at baseline and changes in soluble vascular endothelial growth factor receptor-2 (sVEGFR2) or placental-derived growth factor (PlGF) levels during treatment were explored for their association with outcome.Methods:Twenty-three baseline CAFs, and sVEGFR2 and PlGF changes were measured in 85 and 32 patients, respectively. Associations between baseline CAF levels and efficacy parameters, plus between-week 12 sVEGFR2 and PlGF levels and pazopanib-specific toxicities were investigated.Results:At baseline, low interleukin (IL)-12 p40 subunit and MPC3 levels were associated with better progression-free survival (PFS) at 12 weeks (PFS 12wks), low basic nerve growth factor and hepatocyte growth factor with a better PFS, and low inter-cellular adhesion molecule-1 and IL-2 receptor alpha with prolonged overall survival (OS; all P=0.05). Pazopanib decreased sVEGFR2 and increased PlGF levels. Low sVEGFR2 and high PlGF levels at week 12 were associated with higher-grade hypertension, with TSH elevations and with poorer PFS 12wks, and OS (both P=0.05).Conclusion:Several baseline CAFs were related to outcome parameters. Low sVEGFR2 and high PlGF at week 12 associate with several pazopanib-specific toxicities and poorer efficacy. If confirmed, these factors may be used as early markers for response to and toxicity from pazopanib, enabling further individualisation of STS treatment

Double Umbilical Cord Blood Transplantation in High-Risk Hematological Patients: A Phase II Study Focusing on the Mechanism of Graft Predominance

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

CD45RA(+)CCR7(-) CD8 T cells lacking co-stimulatory receptors demonstrate enhanced frequency in peripheral blood of NSCLC patients responding to nivolumab

Background Checkpoint inhibitors have become standard care of treatment for non-small cell lung cancer (NSCLC), yet only a limited fraction of patients experiences durable clinical benefit, highlighting the need for markers to stratify patient populations. Methods To prospectively identify patients showing response to therapy, we have stained peripheral blood samples of NSCLC patients treated with 2nd line nivolumab (n = 71), as well as healthy controls, with multiplex flow cytometry. By doing so, we enumerated 18 immune cell subsets and assessed expression for 28 T cell markers, which was followed by dimensionality reduction as well as rationale-based analyses. Results In patients with a partial response (PR), representing best overall response (BOR) according to RECIST v1.1, the number of CD8 T cells at baseline and during treatment is similar to those of healthy controls, but 2-fold higher than in patients with progressive and stable disease (PD and SD). CD8 T cell populations in PR patients show enhanced frequencies of T effector memory re-expressing CD45RA (TEMRA) cells, as well as T cells that express markers of terminal differentiatio

Blood-based kinase activity profiling: A potential predictor of response to immune checkpoint inhibition in metastatic cancer

Background Many cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome. Methods Here a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation. Results The kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-