Early-phenotype CAR-T cells for the treatment of pediatric cancers.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising approach for the treatment of childhood cancers, particularly high-risk tumors that fail to respond to standard therapies. CAR-T cells have been highly successful in treating some types of hematological malignancies. However, CAR-T cells targeting solid cancers have had limited success so far for multiple reasons, including their poor long-term persistence and proliferation. Evidence is emerging to show that maintaining CAR-T cells in an early, less-differentiated state in vitro results in superior persistence, proliferation, and antitumor effects in vivo. Children are ideal candidates for receiving less-differentiated CAR-T cells, because their peripheral T-cell pool primarily comprises naïve cells that could readily be harvested in large numbers to generate early-phenotype CAR-T cells. Although several studies have reported different approaches to successfully generate early CAR-T cells, there are only a few clinical trials testing these in adult patients. No trials are currently testing early CAR-T cells in children. Here, we summarize the different strategies used to maintain CAR-T cells in an early phenotypic stage and present evidence suggesting that this approach may be particularly relevant to treating childhood cancers.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Identification of an excellent prognosis subset of human papillomavirus-associated oropharyngeal cancer patients by quantification of intratumoral CD103+ immune cell abundance.

BACKGROUND: Accurate prognostic stratification of human papillomavirus-associated oropharyngeal cancers (HPV+OPSCC) is required to identify patients potentially suitable for treatment deintensification. We evaluated the prognostic significance of CD103, a surface marker associated with tissue-resident memory T cells (TRMs), in two independent cohorts of patients with HPV+OPSCC. PATIENTS AND METHODS: The abundance and distribution of CD103+ immune cells were quantified using immunohistochemistry in a cohort of 189 HPV+OPSCC patients treated with curative intent and correlated with outcome. Findings were then validated in an independent cohort comprising 177 HPV+OPSCCs using univariable and multivariable analysis. Intratumoral CD103+ immune cells were characterized by multispectral fluorescence immunohistochemistry and gene expression analysis. RESULTS: High intratumoral abundance of CD103+ immune cells using a ≥30% cut-off was found in 19.8% of tumors in the training cohort of HPV+OPSCC patients and associated with excellent prognosis for overall survival (OS) with adjusted hazard ratio (HR) of 0.13 [95% confidence interval (CI) 0.02-0.94, P = 0.004]. In the independent cohort of HPV+OPSCCs, 20.4% had high intratumoral CD103+ abundance and an adjusted HR for OS of 0.16 (95% CI 0.02-1.22, P = 0.02). Five year OS of patients with high intratumoral CD103 was 100% across both cohorts. The C-statistic for the multivariate prognostic model with stage and age was significantly improved in both cohorts with the addition of intratumoral CD103+ cell abundance. On the basis of spatial location, co-expression of CD8 and CD69, and gene expression profiles, intratumoral CD103+ cells were consistent with TRMs. CONCLUSION: Quantification of intratumoral CD103+ immune cell abundance provides prognostic information beyond that provided by clinical parameters such as TNM-staging, identifying a population of low risk HPV+OPSCC patients who are good candidates for trials of deintensification strategies.

CD3bright signals on γδ T cells identify IL-17A-producing Vγ6Vδ1+ T cells.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1ß. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.

Natural killer T cell defects in multiple myeloma and the impact of lenalidomide therapy.

The causes of multiple myeloma (MM) remain obscure and there are few known risk factors; however, natural killer T (NKT) cell abnormalities have been reported in patients with MM, and therapeutic targeting of NKT cells is promoted as a potential treatment. We characterized NKT cell defects in treated and untreated patients with MM and determined the impact of lenalidomide therapy on the NKT cell pool. Lenalidomide is an immunomodulatory drug with co-stimulatory effects on NKT cells in vitro and is an approved treatment for MM, although its mode of action in that context is not well defined. We find that patients with relapsed/progressive MM had a marked deficiency in NKT cell numbers. In contrast, newly diagnosed patients had relatively normal NKT cell frequency and function prior to treatment, although a specific NKT cell deficiency emerged after high-dose melphalan and autologous stem cell transplantation (ASCT) regimen. This also impacted NK cells and conventional T cells, but the recovery of NKT cells was considerably delayed, resulting in a prolonged, treatment-induced NKT cell deficit. Longitudinal analysis of individual patients revealed that lenalidomide therapy had no in-vivo impact on NKT cell numbers or cytokine production, either as induction therapy, or as maintenance therapy following ASCT, indicating that its clinical benefits in this setting are independent of NKT cell modulation.

Innate immunity: Looking beyond T-cells in radiation and immunotherapy combinations.

Radiation therapy is an established and effective anti-cancer treatment modality. Extensive pre-clinical experimentation has demonstrated that the pro-inflammatory properties of irradiation may be synergistic with checkpoint immunotherapy. Radiation induces double-stranded DNA breaks (dsDNA). Sensing of the dsDNA activates the cGAS/STING pathway, producing Type 1 interferons essential to recruiting antigen-presenting cells (APCs). Radiation promotes cytotoxic CD8 T-cell recruitment by releasing tumour-associated antigens captured and cross-presented by surveying antigen-presenting cells. Radiation-induced vascular normalisation may further promote T-cell trafficking and drug delivery. Radiation is also immunosuppressive. Recruitment of regulatory T cells (Tregs) and innate cells such as myeloid-derived suppressive cells (m-MDSCs) all counteract the immunostimulatory properties of radiation. Many innate immune cell types operate at the interface of the adaptive immune response. Innate immune cells, such as m-MDSCs, can exert their immunosuppressive effects by expressing immune checkpoints such as PD-L1, further highlighting the potential of combined radiation and checkpoint immunotherapy. Several early-phase clinical studies investigating the combination of radiation and immunotherapy have been disappointing. A greater appreciation of radiotherapy's impact on the innate immune system is essential to optimise radioimmunotherapy combinations. This review will summarise the impact of radiotherapy on crucial cells of the innate immune system and vital immunosuppressive cytokines.

Low-dose carboplatin modifies the tumor microenvironment to augment CAR T cell efficacy in human prostate cancer models.

Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.

Ex vivo culture of chimeric antigen receptor T cells generates functional CD8+ T cells with effector and central memory-like phenotype.

The anti-tumor efficacy of adoptively transferred T cells requires their in vivo persistence and memory polarization. It is unknown if human chimeric antigen receptor (CAR)-expressing T cells can also undergo memory polarization. We examined the functional status of CAR CD8(+) T cells, re-directed to Lewis Y antigen (LeY-T), throughout a period of ex vivo expansion. Immediately before culture CD8(+) T cells comprised a mixture of phenotypes including naive (CD45RA(+)/CCR7(+)/CD27(+)/CD28(+)/perforin-), central memory (CM, CD45RA(-)/CCR7(lo)/CD27(+)/CD28(+)/perforin(lo)), effector memory (EM, CD45RA(-)/CCR7(-)/CD27(+)/CD28(+)/perforin(mod)) and effector (Eff, CD45RA(+)/CCR7(-)/CD27(-)/CD28(-)/perforin(hi)) cells. After transduction and expansion culture of peripheral blood mononuclear cells from normal donors or multiple myeloma patients, CD8(+) LeY-T cells polarized to EM- and CM-like phenotype. CD8(+) LeY-T cells differed from starting CD8(+) CM and EM T cells in that CD27, but not CD28, was downregulated. In addition, CD8(+) LeY-T cells expressed high levels of perforin, similar to starting CD8(+) Eff. CD8(+) LeY-T cells also showed hallmarks of both memory and Eff function, underwent homeostatic proliferation in response to interleukin (IL)-15, and showed interferon (IFN)-γ production and cytotoxicity in response to Le-Y antigen on OVCAR-3 (human ovarian adenocarcinoma) cells. This study confirms CD8(+) LeY-T cells have a CM- and EM-like phenotype and heterogeneous function consistent with potential to persist in vivo after adoptive transfer.

Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen.

We have evaluated the carbohydrate antigen Lewis(Y) (Le(Y)) as a potential target for T-cell immunotherapy of hematological neoplasias. Analysis of 81 primary bone marrow samples revealed moderate Le(Y) expression on plasma cells of myeloma patients and myeloblasts of patients with acute myeloid leukemia (AML) (52 and 46% of cases, respectively). We developed a retroviral vector construct encoding a chimeric T-cell receptor that recognizes the Le(Y) antigen in a major histocompatibility complex-independent manner and delivers co-stimulatory signals to achieve T-cell activation. We have shown efficient transduction of peripheral blood-derived T cells with this construct, resulting in antigen-restricted interferon-gamma secretion and cell lysis of Le(Y)-expressing tumor cells. In vivo activity of gene-modified T cells was demonstrated in the delayed growth of myeloma xenografts in NOD/SCID mice, which prolonged survival. Therefore, targeting Le(Y)-positive malignant cells with T cells expressing a chimeric receptor recognizing Le(Y) was effective both in vitro and in a myeloma mouse model. Consequently, we plan to use T cells manufactured under Good Manufacturing Practice conditions in a phase I immunotherapy study for patients with Le(Y)-positive myeloma or AML.

Absence of retroviral vector-mediated transformation of gene-modified T cells after long-term engraftment in mice.

There is considerable concern regarding the transforming potential of retroviral vectors currently used for gene therapy, with evidence that retroviral integration can lead to leukemia in recipients of gene-modified stem cells. However, it is not clear whether retroviral-mediated transduction of T cells can lead to malignancy. We transduced mouse T cells with a Moloney murine retroviral gene construct and transferred them into congenic mice, which were preconditioned to enhance the engraftment of transferred T cells. Recipients were then observed long-term for evidence of cancer. Transferred T cells persisted in mice throughout life at levels up to 17% with gene copy numbers up to 5.89 x 10(5) per million splenocytes. Mice receiving gene-modified T cells developed tumors at a similar rate as control mice that did not receive T cells, and tumors in both groups of mice were of a similar range of histologies. Hematological malignancies comprised approximately 60% of cancers, and the remaining cancers consisted largely of carcinomas. Importantly, the incidence of lymphomas was similar in both groups of mice, and no lymphomas were found to be of donor T-cell origin. This study indicates that the use of retroviral vectors to transduce T cells does not lead to malignant transformation.

Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

Characterization of activated lymphocyte-tumor cell adhesion.

This study demonstrates the variable expression of ICAM-1 and leukocyte function antigen-3 (LFA-3) on four tumor cell lines (COLO526, K562, Daudi, and HT-29). In addition, phorbol ester (PMA) activation of lymphocytes modulated LFA-1 from a uniform to a clustered surface distribution; whereas after treatment with high levels of Mg2+ ions, the unique epitope for high-affinity LFA-1 was identified using clone Mab24. Using a flow cytometric adhesion assay it was demonstrated that PMA-activated lymphocytes formed conjugates with COLO526 and Daudi, and that these conjugates were inhibited by anti-CD2 with varying inhibition by LFA-1 clones MHM24 and 25.3.1. When lymphocytes were induced to express the high-affinity form of LFA-1, conjugates were identified with COLO526, K562, and Daudi and these conjugates were sensitive to the presence of both CD2 and LFA-1 antibodies. Further studies using confocal microscopy confirmed significant adhesion between peripheral blood lymphocytes pretreated with either PMA or high levels of Mg2+ and the adherent cell line COLO526. In conclusion, this unique study has demonstrated for the first time the important role of the active form of LFA-1 on the lymphocyte cell surface for conjugate formation with an ICAM-1-expressing tumor cell; also, two pathways of cell signaling were identified for conjugate formation to occur.

The binding of proteinase 3 antineutrophil cytoplasmic antibodies (PR3-ANCA) varies in different ELISAs.

BACKGROUND: The demonstration of proteinase 3 specific antineutrophil cytoplasmic antibodies (PR3-ANCA), and the estimation of antibody values are useful in the diagnosis and management of patients with Wegener's granulomatosis (WG). However, external quality assessment programmes suggest that PR3-ANCA binding varies in different assays. AIM: To demonstrate variations in PR3-ANCA binding in different commercial and in house enzyme linked immunosorbent assays (ELISAs). METHOD: Binding of a PR3-ANCA standard and 19 sera from patients with WG was compared in eight commercial and in house assays. Binding was expressed in different units depending on the kit. RESULTS: One commercial assay performed unsatisfactorily. Three commercial kits produced PR3-ANCA binding (70, 102, and 84 U/ml) close to the expected value for the standard (100 U/ml). Serial dilutions of this standard were linear in only one commercial assay and the in house assay. Sera from patients with WG with borderline binding in the in house assay bound in the eight commercial kits at 0-148 kit units; low binding sera ranged from 0 to 273 units; moderately strong sera bound at 7-260 units; and strongly binding sera bound at 13-336 units. In four assays, at least one strongly positive serum bound at levels greater than the provided range. CONCLUSIONS: Levels of antibody binding and units of binding have not been standardised in commercially available PR3-ANCA ELISAs. This may affect the diagnosis and management of patients with WG, in addition to the implementation of international guidelines for treatment.

Acquired protein S deficiency in a patient with systemic lupus erythematosus causing central retinal vein thrombosis.

A 16 year old girl with systemic lupus erythematosus (SLE) developed the rare complication of central retinal vein occlusion. Although classically a disease of older patients, it has been recognised in association with SLE but only in the presence of the lupus anticoagulant or antiphospholipid antibodies. The thrombosis occurred when free protein S concentrations were transiently reduced and there was no family history or other known causes of reduced protein S concentrations. No other prothrombotic risk factors were present.

Antithyroid and antiadrenal autoantibodies in antiglomerular basement membrane disease, thin basement membrane disease and Alport syndrome.

The basement membranes of the glomerulus, thyroid and adrenal all contain the Goodpasture antigen, the target of autoantibodies in antiglomerular basement membrane (GBM) disease. Antithyroid antibodies can be associated with antiGBM disease, and there have been occasional reports of antithyroid antibodies in Alport syndrome, an inherited kidney disease where the GBM lacks the Goodpasture antigen. The aim of this study was to determine how often antithyroid and antiadrenal autoantibodies occurred in antiGBM disease, Alport syndrome and a related condition, thin basement membrane disease (TBMD). Sera from patients with antiGBM disease (n = 19), Alport syndrome (n = 5) or TBMD (n = 13) were tested for antithyroglobulin, antithyroid microsomal and antiadrenal antibodies. Five of the patients with antiGBM disease (5/19, 26%, P NS) had antimicrosomal, and one had antithyroglobulin, antibodies (1/19, 5%, P NS). No patient with Alport syndrome had antithyroid antibodies. One with TBMD (1/13, 8%, P NS) had antithyroglobulin and antimicrosomal antibodies at titres of 1/400 and 1/25,600, respectively. Both patients with antithyroglobulin antibodies had previously been diagnosed with hypothyroidism. No one with antiGBM disease, Alport syndrome or TBMD had antiadrenal antibodies. Antithyroid microsomal antibodies do not occur significantly more often in patients with antiGBM disease than in normals, and antithyroid and antiadrenal antibodies are not associated with Alport syndrome or TBMD.

Mechanism of action of immunomodulatory drugs (IMiDS) in multiple myeloma.

Immunomodulatory drugs (IMiDs) are thalidomide analogues, which possess pleiotropic anti-myeloma properties including immune-modulation, anti-angiogenic, anti-inflammatory and anti-proliferative effects. Their development was facilitated by an improved understanding in myeloma (MM) biology and initiated a profound shift in the therapeutic approach towards MM. Despite the diverse effects of IMiDs in vitro, the relative contribution of each effect towards their ultimate anti-MM activity is still unclear. Based on in vitro data, it appears that anti-proliferative effects and downregulation of crucial cytokines are their most important anti-MM attributes. Although the co-stimulatory effects on T and NK cells have been heralded as a unique and important property of IMiDs towards enhancing anti-MM immune activity, these in vitro effects have yet to be firmly corroborated in vivo. Much is yet to be elucidated regarding the complex interplay of immunomodulatory cytokines that occurs in vivo, which ultimately dictates the net effects of IMiDs in MM-the understanding of which is necessary to facilitate optimal manipulation of these drugs in future MM management.

Testing the NKT cell hypothesis in lenalidomide-treated myelodysplastic syndrome patients.

Myelodysplastic syndrome (MDS) comprises a group of clonal bone marrow disorders characterized by ineffective hematopoiesis and increased predisposition to acute myeloid leukemia. The causes of MDS remain poorly defined, but several studies have reported the NKT cell compartment of patients with MDS is deficient in number and functionally defective. In support of a central role for NKT cells, a pilot clinical study reported that lenalidomide (an approved treatment for MDS) increased NKT cell numbers in patients with MDS, and several in vitro studies showed lenalidomide specifically promoted NKT cell proliferation and cytokine production. We tested this in a much larger study and confirm a moderate in vitro augmentation of some NKT cell functions by lenalidomide, but find no impact on the NKT cell compartment of patients treated with lenalidomide, despite a consistently positive clinical response. We further show that the frequency and cytokine production of NKT cells is normal in patients with MDS before treatment and remains stable throughout 10 months of lenalidomide therapy. Collectively, our data challenge the concept that NKT cell defects contribute to the development of MDS, and show that a clinical response to lenalidomide is not dependent on modulation of NKT cell frequency or function.

A new multi-parameter flow cytometric assay for monitoring lymphoma growth and spread in a pre-clinical murine model for human lymphoma.

BACKGROUND: Mouse survival is commonly used as an indirect measure of lymphoma tumor response to anti-idiotype vaccine; however, this gives no information regarding residual lymphoma cells at primary or metastatic sites. We aimed to develop a method with which to monitor lymphoma tumor kinetics in the mouse as an independent measure of vaccine efficacy. METHODS: We developed a multi-parameter flow cytometric (MPFC) assay for 38C13 mouse lymphoma cells using sequential gating to detect aberrant antigen expression and binding by an anti-idiotype antibody (S1C5). Subsequently, we tested the utility of the MPFC assay in a 38C13 tumor modeling study in the C3H/HeN mouse. RESULTS: The MPFC assay was demonstrated in vitro to have both high specificity and sensitivity for 38C13 lymphoma cells. In tumor kinetic studies in the C3H/HeN mouse, as the tumor enlarged, the MPFC assay showed increasing prevalence of 38C13 cells at the inoculation site in addition to metastases to lymphoid organs and bone marrow. The latter findings were confirmed by both histology and immunohistochemistry. CONCLUSIONS: The MPFC assay is an independent parameter for monitoring 38C13 lymphoma kinetics and could be used to monitor tumor response to individual vaccines or other lymphoma therapy prior to clinical trials.