Hauke Winter

Divergent Roles for CD4+ T Cells in the Priming and Effector/Memory Phases of Adoptive Immunotherapy

The requirement for CD4+ Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4+ T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II β-chain (MHC II KO), revealed that priming of therapeutic CD8+ effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4+ T cell help. The adoptive transfer of CD8+ effector T cells, but not CD4+ effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8+ effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8+ T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8+ T cells were resistant to tumor challenge. Resistance was mediated by CD8+ T cells in mice at 50 days, while both CD4+ and CD8+ T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4+ Th cells are not required for the priming phase of CD8+ effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.

Immunotherapy of Melanoma: A Dichotomy in the Requirement for IFN-γ in Vaccine-Induced Antitumor Immunity Versus Adoptive Immunotherapy

The mechanism by which tumors are rejected following the adoptive transfer of tumor-specific T cells is not well characterized. Recent work has challenged the requirement for cytotoxicity mediated by either the perforin/granzyme or Fas/Fas ligand pathway in T cell-mediated tumor regression. Many reports, including ours, suggest that tumor-specific production of IFN-γ is critical for T cell-mediated tumor regression. However, in most of these studies the evidence to support the role for IFN-γ is only indirect. We have directly examined the requirement for IFN-γ using IFN-γ knockout (GKO) mice. The results show an interesting dichotomy in the requirement for IFN-γ: Antitumor immunity induced by active-specific immunotherapy (vaccination) required IFN-γ, whereas adoptive immunotherapy did not. In GKO mice vaccination with the GM-CSF gene-modified B16BL6-D5 tumor (D5-G6) failed to induce protective immunity against parental D5 tumor. However, adoptive transfer of effector T cells from GKO mice cured 100% of GKO mice with established pulmonary metastases and induced long term antitumor immunity and depigmentation of skin. Furthermore, in vivo neutralization of IFN-γ by mAb treatment or adoptive transfer into IFN-γ receptor knockout mice failed to block the therapeutic efficacy of effector T cells generated from wild-type or perforin knockout mice. Analysis of regressing metastases revealed similar infiltrates of macrophages and granulocytes in both wild-type and GKO mice. These results indicate that in this adoptive immunotherapy model, neither a direct effect on the tumor nor an indirect effect of IFN-γ through activation of myeloid or lymphoid cells is critical for therapeutic efficacy.

Tumour-induced polarization of tumour vaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies.

Previously we have shown that vaccination with the poorly immunogenic B16BL6‐D5 melanoma (D5) elicits a dominant type 2 (T2) cytokine response that fails to protect the host from a subsequent tumour challenge. Here we investigated whether the inherent immunogenicity of a tumour can be correlated with its ability to bias the anti‐tumour cytokine response towards either a type 1 (T1) or a T2 profile. The immune response to six tumours of different inherent immunogenicity was assayed. By isolating l‐selectinlow T cells from tumour vaccine draining lymph nodes (TVDLN), it was possible to detect tumour‐specific cytokine responses from both immunogenic, poorly immunogenic and non‐immunogenic tumours. Immunogenic tumours (MCA‐304, MCA‐309, MPR‐4) induced a predominant tumour‐specific T1 cytokine response. In contrast, weakly (MCA‐310, MPR‐3) and poorly/non‐immunogenic tumours (MPR‐5, D5) sensitized T cells with a predominant tumour‐specific T2 cytokine response. A significant correlation (Pu2003<u20030·025) between immunogenicity and the ratio of tumour‐specific interferon‐γu2003:u2003interleukin‐4 (IL‐4) secretion by TVDLN T cells was identified. We then documented that non‐therapeutic T cells primed by the poorly immunogenic D5, recognized ‘tumour‐rejection’ antigens and that reprogramming their cytokine response, by in vitro culture with IL‐12 and anti‐IL‐4, to a T1 profile uncovered therapeutic efficacy. In contrast, TVDLN T cells primed by a therapeutic vaccine lose therapeutic efficacy when cultured with IL‐4. These results provide insights into the development of a protective anti‐tumour immune response and strengthen the hypothesis that a T1 cytokine response is critical for T‐cell‐mediated tumour regression.

CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells

Previously, we have shown that priming of therapeutic CD8+ T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8+ T cells. Priming of therapeutic CD8+ T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-βR, or receptor activator of NF-κB was not essential. Thus, cross-priming of therapeutic CD8+ T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.

Bronchial Stump Coverage and Postpneumonectomy Bronchopleural Fistula

To prevent postpneumonectomy bronchopleural fistula, coverage of the bronchial stump is recommended, especially for patients treated with neoadjuvant and adjuvant chemotherapy or radiochemotherapy. We compared outcomes after proximal pericardial fat pad coverage and coverage with pleura and surrounding tissues, by retrospective analysis of the records of 243 patients. Postpneumonectomy bronchopleural fistula occurred in 7/143 (4.9%) patients who had pericardial fat pad coverage, and in 6/100 (6.0%) treated by pleural covering. Bronchopleural fistula occurred in 11 patients within 21 days, in one after 2 months, and one after 6 months. Univariate analysis of comorbidities and risk factors did not show any significant differences between the groups. Advanced T stage and carcinomatous lymphangiosis at the resection margin were associated with a higher risk of bronchopleural fistula development, independent of the technique. Reinforcement of the bronchial stump by proximal pericardial fat pad coverage appears to be safe and feasible. It is comparable to coverage with pleura and surrounding tissues.

Lysis of cancer cells by autologous T cells in breast cancer pleural effusates treated with anti-EpCAM BiTE antibody MT110

In the present study, the efficacy of a new drug, i.e. the bispecific single-chain antibody MT110 targeting the epithelial antigen EpCAM and the T-cell antigen CD3 was tested exxa0vivo in malignant pleural effusions (MPEs). EpCAM+ epithelial cells were found in 78% of the MPEs (nxa0=xa018). Exxa0vivo treatment of seven MPEs resulted in a dose-dependent specific lysis of 37xa0±xa027% (±SD) EpCAM+ cells with 10xa0ng/ml (Pxa0=xa00.03) and 57xa0±xa029.5% EpCAM+ cells with 1,000xa0ng/ml MT110 (Pxa0=xa00.016) after 72xa0h. As a prerequisite for redirected lysis, stimulation of the autologous CD4+ and CD8+ cells in MPE by 1,000xa0ng/ml MT110 resulted in 21xa0±xa017% CD4+/CD25+ and 29.4xa0±xa022% CD8+/CD25+ cells (Pxa0=xa00.016, respectively) after 72xa0h. This was confirmed by a 22-fold release of TNF-α and 230-fold release of IFN-γ (1,000xa0ng/ml, 48xa0h, Pxa0=xa00.03, respectively). Thus, relapsed breast cancer patients resistant to standard treatment might benefit from targeted therapy using MT110.

Immunotherapy of Lung Cancer: An Update

In Germany lung cancer is the leading cause of cancerassociated death in men. Surgery, chemotherapy and radiation may enhance survival of patients suffering from lung cancer but the enhancement is typically transient and mostly absent with advanced disease; eventually more than 90% of lung cancer patients will die of disease. New approaches to the treatment of lung cancer are urgently needed. Immunotherapy may represent one new approach with low toxicity and high specificity but implementation has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape immune responses. Several different immunotherapeutic treatment strategies have been developed. This review examines the current state of development and recent advances with respect to non-specific immune stimulation, cellular immunotherapy (specific and non-specific), therapeutic cancer vaccines and gene therapy for lung cancer. The focus is primarily placed on immunotherapeutic cancer treatments that are already in clinical trial or well progressed in preclinical studies. Although there seems to be a promising future for immunotherapy in lung cancer, presently there is not standard immunotherapy available for clinical routine.

Immunotherapy of Cancer: Key Findings and Commentary on the Third Tegernsee Conference

Cancer immunotherapy broadly includes active immunization, as in the use of cancer vaccines, passive immunization, such as the use of adoptive cell therapy and antibodies that modulate tumor function, and immunostimulation, using antibodies and small molecules to treat malignancy by activating or unleashing an endogenous immune response against tumor cells. Currently, >100 different monoclonal antibodies are in use or under evaluation for use as therapeutic agents in various malignancies. Active stimulation of the hosts immune system holds promise for achieving durable remission of malignant disease and represents a nontoxic method of therapy if tumor-specific effector cells can be selectively targeted. However, no active-specific treatment strategy (i.e., a therapeutic cancer vaccine) has yet found its way into the clinical armamentarium, although several promising recent reports suggest that, for follicular lymphoma, prostate cancer, and melanoma, clinical benefit was shown for the first time in randomized trials with a vaccine approach. Here, we report on the key findings of the Third Tegernsee Conference on Immunotherapy of Cancer (Feldafing, Germany, July 2-4, 2009) and provide short commentaries on data presented at this meeting regarding the future role of cancer vaccines, recent developments in adoptive cellular therapy, ways to improve immunotherapeutic treatment modalities (e.g., by manipulating the tumor microenvironment), and some novel targeted therapies that are well advanced in clinical testing, all of which have implications for future oncology practice.

Comparing Azole Plasma Trough Levels in Lung Transplant Recipients: Percentage of Therapeutic Levels and Intrapatient Variability

Background: This study compared therapeutic azole plasma trough levels (APL) of the azole antimycotics itraconazole (ITR), voriconazole (VOR), and posaconazole (POS) in lung transplant recipients and analyzed the influencing factors. In addition, intrapatient variability for each azole was determined. Methods: From July 2012 to July 2015, 806 APL of ITR, VOR, posaconazole liquid (POS-Liq), and posaconazole tablets (POS-Tab) were measured in 173 patients of the Munich Lung Transplantation Program. Therapeutic APL were defined as follows: ITR, ≥700 ng/mL; VOR, 1000–5500 ng/mL; and POS, ≥700 ng/mL (prophylaxis) and ≥1000 ng/mL (therapy). Results: VOR and POS-Tab reached the highest number of therapeutic APL, whereas POS-Liq showed the lowest percentage (therapy: ITR 50%, VOR 70%, POS-Liq 38%, and POS-Tab 82%; prophylaxis: ITR 62%, VOR 85%, POS-Liq 49%, and POS-Tab 76%). Risk factors for subtherapeutic APL of all azoles were the azole dose (ITR, P < 0.001; VOR, P = 0.002; POS-Liq, P = 0.006) and age over 60 years (ITR, P = 0.003; VOR, P = 0.002; POS-Liq, P = 0.039; POS-Tab, P < 0.001). Cystic fibrosis was a significant risk factor for subtherapeutic APL for VOR and POS-Tab (VOR, P = 0.002; POS-Tab, P = 0.005). Double lung transplantation (LTx) was significantly associated with less therapeutic APL for VOR and POS-Liq (VOR, P = 0.030; POS-Liq, P < 0.001). Concomitant therapy with 80 mg pantoprazole led to significantly fewer therapeutic POS APL as compared to 40 mg (POS-Liq, P = 0.015; POS-Tab, P < 0.001). VOR displayed the greatest intrapatient variability (46%), whereas POS-Tab showed the lowest (32%). Conclusions: Our study showed that VOR and POS-Tab achieve the highest percentage of therapeutic APL in patients with LTx; POS-Tab showed the lowest intrapatient variability. APL are significantly influenced by azole dose, age, cystic fibrosis, type of LTx, and comedication with proton-pump inhibitors. Considering the high number of subtherapeutic APL, therapeutic drug monitoring should be integrated in the post-LTx management.

Extracorporeal Circulation During Lung Transplantation Procedures: A Meta-analysis

Extracorporeal circulation (ECC) is an invaluable tool in lung transplantation (lutx). More than the past years, an increasing number of centers changed their standard for intraoperative ECC from cardiopulmonary bypass (CPB) to extracorporeal membrane oxygenation (ECMO) – with differing results. This meta-analysis reviews the existing evidence. An online literature research on Medline, Embase, and PubMed has been performed. Two persons independently judged the papers using the ACROBAT-NRSI tool of the Cochrane collaboration. Meta-analyses and meta-regressions were used to determine whether veno-arterial ECMO (VA-ECMO) resulted in better outcomes compared with CPB. Six papers – all observational studies without randomization – were included in the analysis. All were considered to have serious bias caused by heparinization as co-intervention. Forest plots showed a beneficial trend of ECMO regarding blood transfusions (packed red blood cells (RBCs) with an average mean difference of −0.46 units [95% CI = −3.72, 2.80], fresh-frozen plasma with an average mean difference of −0.65 units [95% CI = −1.56, 0.25], platelets with an average mean difference of −1.72 units [95% CI = −3.67, 0.23]). Duration of ventilator support with an average mean difference of −2.86 days [95% CI = −11.43, 5.71] and intensive care unit (ICU) length of stay with an average mean difference of −4.79 days [95% CI = −8.17, −1.41] were shorter in ECMO patients. Extracorporeal membrane oxygenation treatment tended to be superior regarding 3 month mortality (odds ratio = 0.46, 95% CI = 0.21–1.02) and 1 year mortality (odds ratio = 0.65, 95% CI = 0.37–1.13). However, only the ICU length of stay reached statistical significance. Meta-regression analyses showed that heterogeneity across studies (sex, year of ECMO implementation, and underlying disease) influenced differences. These data indicate a benefit of the intraoperative use of ECMO as compared with CPB during lung transplant procedures regarding short-term outcome (ICU stay). There was no statistically significant effect regarding blood transfusion needs or long-term outcome. The superiority of ECMO in lutx patients remains to be determined in larger multi-center randomized trials.