The majority of experiments investigating the immune response to gastrointestinal helminth infection use a single bolus infection. However, in situ individuals are repeatedly infected with low doses. Therefore, to model natural infection, mice were repeatedly infected (trickle infection) with low doses of Trichuris muris. Trickle infection resulted in the slow acquisition of immunity reflected by a gradual increase in worm burden followed by partial expulsion. Flow cytometry revealed that the CD4+ T cell response shifted from Th1 dominated to Th2 dominated, which coincided with an increase in Type 2 cytokines. The development of resistance following trickle infection was associated with increased worm expulsion effector mechanisms including goblet cell hyperplasia, Muc5ac production and increased epithelial cell turn over. Depletion of CD4+ T cells reversed resistance confirming their importance in protective immunity following trickle infection. In contrast, depletion of group 2 innate lymphoid cells did not alter protective immunity. T. muris trickle infection resulted in a dysbiotic mircrobiota which began to recover alpha diversity following the development of resistance. These data establish trickle infection as a robust and informative model for analysis of immunity to chronic intestinal helminth infection more akin to that observed under natural infection conditions and confirms the importance of CD4+ T cell adaptive immunity in host protection.

Summary Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

Chimeric antigen receptor (CAR) T cell therapy has achieved unrivalled success in the treatment of B cell and plasma cell malignancies, with five CAR T cell products now approved by the US Food and Drug Administration (FDA). However, CAR T cell therapies for solid tumours have not been nearly as successful, owing to several additional challenges. Here, we discuss mechanisms of tumour resistance in CAR T cell therapy and the emerging strategies that are under development to engineer CAR T cells to overcome resistance.

The circumsporozoite protein (CSP) builds up the surface coat of sporozoites and is the leading malaria pre-erythrocytic-stage vaccine candidate. CSP has been shown to induce robust CD8+ T cell responses that are capable of eliminating developing parasites in hepatocytes resulting in protective immunity. In this study, we characterised the importance of the immunodominant CSP-derived epitope, SYIPSAEKI, of Plasmodium berghei in both sporozoite- and vaccine-induced protection in murine infection models. In BALB/c mice, where SYIPSAEKI is efficiently presented in the context of the major histocompatibility complex class I (MHC-I) molecule H-2-Kd, we established that epitope-specific CD8+ T cell responses contribute to parasite killing following sporozoite immunisation. Yet, sterile protection was achieved in the absence of this epitope substantiating the concept that other antigens can be sufficient for parasite-induced protective immunity. Furthermore, we demonstrated that SYIPSAEKI-specific CD8+ T cell responses elicited by viral-vectored CSP-expressing vaccines effectively targeted parasites in hepatocytes. The resulting sterile protection strictly relied on the expression of SYIPSAEKI. In C57BL/6 mice, which are unable to present the immunodominant epitope, CSP-based vaccines did not confer complete protection, despite the induction of high levels of CSP-specific antibodies. These findings underscore the significance of CSP in protection against malaria pre-erythrocytic stages and demonstrate that a significant proportion of the protection against the parasite is mediated by CD8+ T cells specific for the immunodominant CSP-derived epitope.

21The circumsporozoite protein (CSP) builds up the surface coat of sporozoites and is the leading 22 malaria pre-erythrocytic-stage vaccine candidate. CSP has been shown to induce robust CD8+ 23 T cell responses that are capable of eliminating developing parasites in hepatocytes resulting 24 in protective immunity. In this study, we characterised the importance of the immunodominant 25 CSP-derived epitope, SYIPSAEKI, of Plasmodium berghei in both sporozoite-and vaccine-26 induced protection in murine infection models. In BALB/c mice, where SYIPSAEKI is efficiently 27 presented in the context of the major histocompatibility complex class I (MHC-I) molecule H-28 2-K d , we established that epitope-specific CD8+ T cell responses contribute to parasite killing 29 following sporozoite immunisation. Yet, sterile protection was achieved in the absence of this 30 epitope substantiating the concept that other antigens can be sufficient for parasite-induced 31 protective immunity. Furthermore, we demonstrated that SYIPSAEKI-specific CD8+ T cell 32 responses elicited by viral-vectored CSP-expressing vaccines effectively targeted parasites in 33 hepatocytes. The resulting sterile protection strictly relied on the expression of SYIPSAEKI. In 34 C57BL/6 mice, which are unable to present the immunodominant epitope, CSP-based 35 vaccines did not confer complete protection, despite the induction of high levels of CSP-36 specific antibodies. These findings underscore the significance of CSP in protection against 37 malaria pre-erythrocytic stages and demonstrate that a significant proportion of the protection 38 against the parasite is mediated by CD8+ T cells specific for the immunodominant CSP-derived 39 epitope. 40 41 3 INTRODUCTION 42 Malaria is caused by a protozoan parasite of the genus Plasmodium and remains a major 43 global health challenge in tropical and subtropical countries (1). A vaccine that diminishes the 44 burden of disease and prevents malaria transmission remains a decisive goal for malaria 45 elimination programmes. As a gold standard in malaria vaccination, multiple immunisations of 46 g-radiation-attenuated Plasmodium sporozoites (RAS) can completely protect against wild-47 type (WT) sporozoite challenge (2-4). This parasite-induced protection targets the developing 48 exo-erythrocytic forms in hepatocytes, also called liver stages, and completely abrogates blood 49 stage infection. Antibodies and T cells have been implicated as important mechanisms of 50 protection (5), and CD8+ T cells are the prime mediators of cell-mediated protective immunity, 51 as exemplified in murine (6, 7) and non-human primate (8) infection models. 52 53 The circumsporozoite protein (CSP), the major surface coat protein of the malaria sporozoite, 54 has been at the forefront of vaccination studies -being the basis of RTS,S/AS01, the most 55 progressed malaria vaccine candidate to date (9). Immunisation of BALB/c mice 56 with Plasmodium berghei (Pb) or P. yoelii (Py) RAS evokes immunodominant major 57 histocompatibility complex cla...

13 The majority of experiments investigating the immune response to gastrointestinal helminth 14 infection use a single bolus infection. However, in situ individuals are repeatedly infected with low 15 doses. Therefore, to model natural infection, mice were repeatedly infected (trickle infection) with 16 low doses of Trichuris muris. Trickle infection resulted in the slow acquisition of immunity reflected 17 by a gradual increase in worm burden followed by a partial expulsion. Flow cytometry revealed 18 that the CD4+ T cell response shifted from Th1 dominated to Th2 dominated, which coincided 19 with an increase in Type 2 cytokines. The development of resistance following trickle infection 20 was associated with increased worm expulsion effector mechanisms including goblet cell 21 hyperplasia, Muc5ac production and increased epithelial cell turn over. Depletion of CD4+ T cells 22 reversed resistance confirming their importance in protective immunity following trickle infection. 23In contrast, depletion of group 2 innate lymphoid cells did not alter protective immunity. T. muris 24 trickle infection resulted in a dysbiotic mircrobiota which began to recover alpha diversity following 25 the development of resistance.26 These data support trickle infection as a robust and informative model for analysis of immunity to 27 chronic intestinal helminth infection more akin to that observed under natural infection conditions 28 and confirms the importance of CD4+ T cell adaptive immunity in host protection. 29 30 Author Summary 31 Infection with parasitic worms (helminths) is a considerable cause of morbidity in humans.32 Understanding how we respond to infection is crucial to developing novel therapies. Laboratory 33 models of helminth infection have been a valuable tool in understanding fundamental immune 34 responses to infection. However, typically an individual mouse will be infected with a large, single-35 dose of the parasite. This is in contrast to the natural scenario in which individuals will receive 36 frequent low level exposures. What is unknown is how repeated infection alters the development 37 of immunity to infection. We have developed a laboratory model to tackle this question. We 38 infected mice with the model helminth Trichuris muris on a weekly basis and assessed a range of 39 responses in comparison with a more traditional infection system. We found striking differences 40 in the dynamics of the infection, the host immune response, and in changes to host gut microbial 41 populations. Our study shows how resistance to helminth infection can develop over time in 42 response to repeat infection, and provides a model system that better reflects human immunity to 43 this parasite. 45 Introduction46 Gastrointestinal (GI) dwelling nematodes infect approximately 1 billion people worldwide causing 47 significant ill health (1). Prevalence is high in endemic areas although intensity of infection varies 3 48 with age, suggesting acquired immunity develops, although sterile immunity is rare and individuals 49 ar...