Expansion of endogenous T cells in CSF of pediatric CNS tumor patients undergoing locoregional delivery of IL13R〿2-targeting CAR T cells: an interim analysis.

Outcomes for pediatric brain tumor patients remain poor, and there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. Here, we present interim results from the first six pediatric patients treated on an ongoing phase I clinical trial (NCT04510051) of IL13BBζ-CAR T cells delivered weekly into the lateral cerebral ventricles, identifying clonal expansion of endogenous CAR-negative CD8+ T cells in the cerebrospinal fluid (CSF) over time. Additionally, of the five patients evaluable for disease response, three experienced transient radiographic and/or clinical benefit not meeting protocol criteria for response. The first three patients received CAR T cells alone; later patients received lymphodepletion before the first infusion. There were no dose limiting toxicities (DLTs). Aside from expected cytopenias in patients receiving lymphodepletion, serious adverse events possibly attributed to CAR T cell infusion were limited to one episode of headache and one of liver enzyme elevation. One patient withdrew from treatment during the DLT period due to a Grade 3 catheter-related infection and was not evaluable for disease response, although this was not attributed to CAR T cell infusion. Importantly, scRNA- and scTCR-sequence analyses provided insights into CAR T cell interaction with the endogenous immune system. In particular, clonally expanded endogenous CAR- T cells were recovered from the CSF, but not the peripheral blood, of patients who received intraventricular IL13BBζ-CAR T cell therapy. Additionally, although immune infiltrates in CSF and post-therapy tumor did not generally correlate, a fraction of expanded T cell receptors (TCRs) was seen to overlap between CSF and tumor. This has important implications for what samples are collected on these trials and how they are analyzed. These initial findings provide support for continued investigation into locoregionally-delivered IL13BBζ-CAR T cells for children with brain tumors.

Human Breast Milk: From Food to Active Immune Response With Disease Protection in Infants and Mothers.

Breastfeeding is associated with long-term wellbeing including low risks of infectious diseases and non-communicable diseases such as asthma, cancer, autoimmune diseases and obesity during childhood. In recent years, important advances have been made in understanding the human breast milk (HBM) composition. Breast milk components such as, non-immune and immune cells and bioactive molecules, namely, cytokines/chemokines, lipids, hormones, and enzymes reportedly play many roles in breastfed newborns and in mothers, by diseases protection and shaping the immune system of the newborn. Bioactive components in HBM are also involved in tolerance and appropriate inflammatory response of breastfed infants if necessary. This review summarizes the current literature on the relationship between mother and her infant through breast milk with regard to disease protection. We will shed some light on the mechanisms underlying the roles of breast milk components in the maintenance of health of both child and mother.

Vitamin C, From Supplement to Treatment: A Re-Emerging Adjunct for Cancer Immunotherapy?

Vitamin C (VitC), in addition to its role as a general antioxidant, has long been considered to possess direct anti-cancer activity at high doses. VitC acts through oxidant and epigenetic mechanisms, which at high doses can exert direct killing of tumor cells in vitro and delay tumor growth in vivo. Recently, it has also been shown that pharmacologic-dose VitC can contribute to control of tumors by modulating the immune system, and studies have been done interrogating the role of physiologic-dose VitC on novel adoptive cellular therapies (ACTs). In this review, we discuss the effects of VitC on anti-tumor immune cells, as well as the mechanisms underlying those effects. We address important unanswered questions concerning both VitC and ACTs, and outline challenges and opportunities facing the use of VitC in the clinical setting as an adjunct to immune-based anti-cancer therapies.

Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer.

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.

Correction to: Cancer immunotherapy with γδ T cells: many paths ahead of us.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cancer immunotherapy with γδ T cells: many paths ahead of us.

γδ T cells play uniquely important roles in stress surveillance and immunity for infections and carcinogenesis. Human γδ T cells recognize and kill transformed cells independently of human leukocyte antigen (HLA) restriction, which is an essential feature of conventional αß T cells. Vγ9Vδ2 γδ T cells, which prevail in the peripheral blood of healthy adults, are activated by microbial or endogenous tumor-derived pyrophosphates by a mechanism dependent on butyrophilin molecules. γδ T cells expressing other T cell receptor variable genes, notably Vδ1, are more abundant in mucosal tissue. In addition to the T cell receptor, γδ T cells usually express activating natural killer (NK) receptors, such as NKp30, NKp44, or NKG2D which binds to stress-inducible surface molecules that are absent on healthy cells but are frequently expressed on malignant cells. Therefore, γδ T cells are endowed with at least two independent recognition systems to sense tumor cells and to initiate anticancer effector mechanisms, including cytokine production and cytotoxicity. In view of their HLA-independent potent antitumor activity, there has been increasing interest in translating the unique potential of γδ T cells into innovative cellular cancer immunotherapies. Here, we discuss recent developments to enhance the efficacy of γδ T cell-based immunotherapy. This includes strategies for in vivo activation and tumor-targeting of γδ T cells, the optimization of in vitro expansion protocols, and the development of gene-modified γδ T cells. It is equally important to consider potential synergisms with other therapeutic strategies, notably checkpoint inhibitors, chemotherapy, or the (local) activation of innate immunity.

Vitamin C supports conversion of human γδ T cells into FOXP3-expressing regulatory cells by epigenetic regulation.

Human γδ T cells are potent cytotoxic effector cells, produce a variety of cytokines, and can acquire regulatory activity. Induction of FOXP3, the key transcription factor of regulatory T cells (Treg), by TGF-ß in human Vγ9 Vδ2 T cells has been previously reported. Vitamin C is an antioxidant and acts as multiplier of DNA hydroxymethylation. Here we have investigated the effect of the more stable phospho-modified Vitamin C (pVC) on TGF-ß-induced FOXP3 expression and the resulting regulatory activity of highly purified human Vγ9 Vδ2 T cells. pVC significantly increased the TGF-ß-induced FOXP3 expression and stability and also increased the suppressive activity of Vγ9 Vδ2 T cells. Importantly, pVC induced hypomethylation of the Treg-specific demethylated region (TSDR) in the FOXP3 gene. Genome-wide methylation analysis by Reduced Representation Bisulfite Sequencing additionally revealed differentially methylated regions in several important genes upon pVC treatment of γδ T cells. While Vitamin C also enhances effector functions of Vγ9 Vδ2 T cells in the absence of TGF-ß, our results demonstrate that pVC potently increases the suppressive activity and FOXP3 expression in TGF-ß-treated Vγ9 Vδ2 T cells by epigenetic modification of the FOXP3 gene.

A comparative view on vitamin C effects on αß- versus γδ T-cell activation and differentiation.

Vitamin C (VitC) is an essential vitamin that needs to be provided through exogenous sources. It is a potent anti-oxidant, and an essential cofactor for many enzymes including a group of enzymes that modulate epigenetic regulation of gene expression. Moreover, VitC has a significant influence on T-cell differentiation, and can directly interfere with T-cell signaling. Conventional CD4 and CD8 T cells express the αß TCR and recognize peptide antigens in the context of MHC presentation. The numerically small population of γδ T cells recognizes antigens in an MHC-independent manner. γδ T cells kill a broad variety of malignant cells, and because of their unique features, are interesting candidates for cancer immunotherapy. In this review, we summarize what is known about the influence of VitC on T-cell activation and differentiation with a special focus on γδ T cells. The known mechanisms of action of VitC on αß T cells are discussed and extrapolated to the effects observed on γδ T-cell activation and differentiation. Overall, VitC enhances proliferation and effector functions of γδ T cells and thus may help to increase the efficacy of γδ T cells applied as cancer immunotherapy in adoptive cell transfer.

In vitro expansion of Vγ9Vδ2 T cells for immunotherapy.

Vγ9Vδ2 T cells can exhibit potent anti-tumor activity and have gained much attention as an interesting tool for cancer immunotherapy by adoptive cell transfer. In this chapter, we explain the different approaches for the in vitro expansion of Vγ9Vδ2 T cells for their subsequent adoptive transfer and present a detailed protocol for the successful in vitro expansion at the laboratory scale. Finally, we discuss strategies for optimization and parameters necessary for the adaption to a clinical scale protocol.

Vitamin C promotes the proliferation and effector functions of human γδ T cells.

γδ T cells are of interest as effector cells for cellular immunotherapy due to their HLA-non-restricted lysis of many different tumor cell types. Potential applications include the adoptive transfer of in vitro-expanded γδ T cells. Therefore, it is important to optimize the culture conditions to enable maximal proliferative and functional activity. Vitamin C (L-ascorbic acid) is an essential vitamin with multiple effects on immune cells. It is a cofactor for several enzymes, has antioxidant activity, and is an epigenetic modifier. Here, we investigated the effects of vitamin C (VC) and its more stable derivative, L-ascorbic acid 2-phosphate (pVC), on the proliferation and effector function of human γδ T cells stimulated with zoledronate (ZOL) or synthetic phosphoantigens (pAgs). VC and pVC did not increase γδ T-cell expansion within ZOL- or pAg-stimulated PBMCs, but increased the proliferation of purified γδ T cells and 14-day-expanded γδ T-cell lines in response to γδ T-cell-specific pAgs. VC reduced the apoptosis of γδ T cells during primary stimulation. While pVC did not prevent activation-induced death of pAg-restimulated γδ T cells, it enhanced the cell cycle progression and cellular expansion. Furthermore, VC and pVC enhanced cytokine production during primary activation, as well as upon pAg restimulation of 14-day-expanded γδ T cells. VC and pVC also increased the oxidative respiration and glycolysis of γδ T cells, but stimulus-dependent differences were observed. The modulatory activity of VC and pVC might help to increase the efficacy of γδ T-cell expansion for adoptive immunotherapy.

TGF-ß enhances the cytotoxic activity of Vδ2 T cells.

TGF-ß is a pleiotropic cytokine with multiple roles in immunity. Apart from its suppressive activity, TGF-ß is a driving cytokine in the differentiation of induced regulatory T cells (iTreg) but also in the polarization of interleukin-9 (IL-9) producing T helper 9 (Th9) T cells. Human Vδ2 expressing γδ T cells exert potent cytotoxicity towards a variety of solid tumor and leukemia/lymphoma target cells and thus are in the focus of current strategies to develop cell-based immunotherapies. Here we report that TGF-ß unexpectedly augments the cytotoxic effector activity of short-term expanded Vδ2 T cells when purified γδ T cells are activated with specific pyrophosphate antigens and IL-2 or IL-15 in the presence of TGF-ß. TGF-ß up-regulates the expression of CD54, CD103, interferon-γ, IL-9 and granzyme B in γδ T cells while CD56 and CD11a/CD18 are down-regulated. Moreover, we show that CD103 (αE/ß7 integrin) is recruited to the immunological synapse in γδ T cells. Increased cytotoxic activity of TGF-ß-exposed γδ T cells is reduced by anti-CD103 and further diminished upon additional anti-CD11a antibody treatment, pointing to a role of cellular adhesion in the enhanced cytolytic activity. Furthermore, magnetically sorted CD103-positive Vδ2 T cells exhibit superior cytolytic activity. In view of the importance of CD103 for tissue homing of lymphocytes, our results suggest that adoptive transfer of CD103-expressing Vδ2 T cells might favor their homing to solid tumors.