Lack of B and T cell reactivity towards IDH1R132H in blood and tumor tissue from LGG patients.

PURPOSE: Mutations in the isocitrate dehydrogenase-1 gene (IDH1) occur at high frequency in grade II-III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous affecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specific B and T cell reactivity in blood and tumor tissue of LGG patients. METHODS: Sera from IDH1R132H-mutated LGG patients (n = 27) were assayed for the presence of a neo-specific antibody response using ELISA. In addition, PBMCs (n = 36) and tumor-infiltrating lymphocytes (TILs, n = 10) were measured for T cell activation markers and IFN-γ production by flow cytometry and ELISA. In some assays, frequencies of CD4 T cells specific for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays. RESULTS: Despite high sensitivity of our assay, we failed to detect IDH1R132H-specific IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following pre-enrichment of frequencies of IDH1R132H-specific CD4 T cells. Finally, we did not detect IDH1R132H-specific CD4 T cells among TILs. CONCLUSIONS: The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1R132H is not sufficiently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients.

CD4+ T-cell alloreactivity toward mismatched HLA class II alleles early after double umbilical cord blood transplantation.

Although double umbilical cord blood transplantation (dUCBT) in adult patients may be associated with less graft failure compared with single UCBT, hematopoietic recovery generally originates from a single cord blood unit (CBU). CBU predominance is still incompletely understood. We recently showed that blood CD4+ T-cell numbers rapidly increase after dUCBT, and early CD4+ T-cell chimerism predicts for graft predominance. Given the frequent HLA class II allele mismatches between CBUs in dUCBT, we hypothesized that alloreactive HLA class II-specific CD4+ T cells from the "winning" CBU may contribute to rejection of the "loser" CBU. We evaluated whether CD4+ T cells originating from the predominant (PD)-CBU would recognize HLA class II allele mismatches, expressed by the nonengrafting (NE)-CBU. Alloreactive effector CD4+ T cells toward 1 or more mismatched HLA class II alleles of the NE-CBU were detected in 11 of 11 patients, with reactivity toward 29 of 33 (88%) tested mismatches, and the strongest reactivity toward DR and DQ alleles early after dUCBT. Mismatched HLA class II allele-specific CD4+ T cells recognized primary leukemic cells when the mismatched HLA class II allele was shared between NE-CBU and patient. Our results suggest that cytotoxicity exerted by CD4+ T cells from the PD-CBU drives the rapid rejection of the NE-CBU, whose alloreactive effect might also contribute to graft-versus-leukemia.

Recurrence of melanoma following T cell treatment: continued antigen expression in a tumor that evades T cell recruitment.

Clinical therapy with T cells shows promise for cancer patients, but is currently challenged by incomplete responses and tumor relapse. The exact mechanisms that contribute to tumor relapse remain largely unclear. Here, we treated mouse melanomas with T cell receptor-engineered T cells directed against a human peptide-major histocompatibility complex antigen in immune-competent mice. T cells resulted in significant tumor regression, which was followed by relapse in about 80-90% of mice. Molecular analysis revealed that relapsed tumors harbored nonmutated antigen genes, not silenced by promoter methylation, and functionally expressed surface antigen at levels equal to nontreated tumors. Relapsed tumors resisted a second in vivo T cell treatment, but regained sensitivity to T cell treatment upon retransplantation in mice. Notably, relapsed tumors demonstrated decreased levels of CD8 T cells and monocytes, which were substantiated by downregulated expression of chemoattractants and adhesion molecules. These observations were confirmed when using T cells specific for a less immunogenic, endogenous mouse melanoma antigen. We conclude that tumors, when exposed to T cell treatment, can relapse without loss of antigen and develop a milieu that evades recruitment of effector CD8 T cells. Our findings support the concept to target the tumor milieu to aid T cell therapy in limiting tumor relapse.

TCR-engineered T-cells directed against Ropporin-1 constitute a safe and effective treatment for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) shows an urgent need for new therapies. We discovered Ropporin-1 (ROPN1) as a target to treat TNBC with T-cells. ROPN1 showed high and homogenous expression in 90% of primary and metastatic TNBC but not in healthy tissues. HLA-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCRs) from naïve repertoires. Following gene introduction into T-cells and stringent selection, we retrieved a highly specific TCR directed against the epitope FLYTYIAKV that did not recognize non-cognate epitopes from alternative source proteins. Notably, this TCR mediated killing of three-dimensional tumoroids in vitro and tumor cells in vivo and outperformed standard-of-care drugs. Finally, the T-cell product expressing this TCR and manufactured using a clinical protocol fulfilled standard safety and efficacy assays. Collectively, we have identified and preclinically validated ROPN1 as a target and anti-ROPN1 TCR T-cells as a treatment for the vast majority of TNBC patients.

Gene Engineering T Cells with T-Cell Receptor for Adoptive Therapy.

Prior to clinical testing of adoptive T-cell therapy with T-cell receptor (TCR)-engineered T cells, TCRs need to be retrieved, annotated, gene-transferred, and extensively tested in vitro to accurately assess specificity and sensitivity of target recognition. Here, we present a fundamental series of protocols that cover critical preclinical parameters, thereby enabling the selection of candidate TCRs for clinical testing.

Intratumoral Niches of B Cells and Follicular Helper T Cells, and the Absence of Regulatory T Cells, Associate with Longer Survival in Early-Stage Oral Tongue Cancer Patients.

In early oral squamous cell carcinoma (OSCC), the occurrence of clusters between CD20 B cells and CD4 T cells in the invasive margin (IM) can be captured by using the CD20 cluster score, and is positively associated with patient survival. However, the exact contribution of different CD4 T cell subsets, as well as B cell subsets toward patient prognosis is largely unknown. To this end, we studied regulatory T cells ((Treg cells) FOXP3 and CD4), T helper-type 1 cells ((Th1 cells) Tbet and CD4), follicular helper T cells ((Tfh cells) Bcl6 and CD4), B cells (CD20), germinal center B cells ((GC B cells) BCL6 and CD20), and follicular dendritic cells ((fDCs) CD21) for their density, location, and interspacing using multiplex in situ immunofluorescence of 75 treatment-naïve, primary OSCC patients. We observed that Treg, Th1-, Tfh-, and GC B cells, but not fDCs, were abundantly present in the stroma as compared with the tumor, and in the IM as compared with in the center of the tumor. Patients with high CD20 cluster scores had a high density of all three CD4 T cell subsets and GC B cells in the stromal IM as compared with patients with low CD20 cluster scores. Notably, enriched abundance of Tfh cells (HR 0.20, p = 0.04), and diminished abundance of Treg cells (HR 0.10, p = 0.03), together with an overall short distance between Tfh and B cells (HR:0.08, p < 0.01), but not between Treg and B cells (HR 0.43, p = 0.28), were significantly associated with overall survival of patients with OSCC. Our study identified the prognostic value of clusters between CD20 B cells and Tfh cells in the stromal IM of OSCC patients, and enabled an improved understanding of the clinical value of a high CD20 cluster score, which requires validation in larger clinical cohorts.

Anti-PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8+ T cells.

PURPOSE: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti-PD-1 efficacy in patients with mUC. EXPERIMENTAL DESIGN: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease). RESULTS: In responders, baseline fractions of CD4+ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1+ CD4+ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB+ CD28+ CD4+ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet+ CD4+ T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8+ T cells, CD11b+ myeloid cells, and tumor cells. CONCLUSIONS: A decrease in the fraction of circulating PD-1+ CD4+ T cells, and juxtaposition of Th1, CD8+, and myeloid cells was associated with response to anti-PD-1 treatment in patients with mUC.

V-Domain Ig Suppressor of T Cell Activation (VISTA) Expression Is an Independent Prognostic Factor in Multiple Myeloma.

Multiple myeloma (MM) is characterized by loss of anti-tumor T cell immunity. Despite moderate success of treatment with anti-PD1 antibodies, effective treatment is still challenged by poor T cell-mediated control of MM. To better enable identification of shortcomings in T-cell immunity that relate to overall survival (OS), we interrogated transcriptomic data of bone marrow samples from eight clinical trials (n = 1654) and one trial-independent patient cohort (n = 718) for multivariate analysis. Gene expression of V-domain Ig suppressor of T cell activation (VISTA) was observed to correlate to OS [hazard ratio (HR): 0.72; 95% CI: 0.61-0.83; p = 0.005]. Upon imaging the immune contexture of MM bone marrow tissues (n = 22) via multiplex in situ stainings, we demonstrated that VISTA was expressed predominantly by CD11b+ myeloid cells. The combination of abundance of VISTA+, CD11b+ cells in the tumor but not stromal tissue together with low presence of CD8+ T cells in the same tissue compartment, termed a high VISTA-associated T cell exclusion score, was significantly associated with short OS [HR: 16.6; 95% CI: 4.54-62.50; p < 0.0001]. Taken together, the prognostic value of a combined score of VISTA+, CD11b+ and CD8+ cells in the tumor compartment could potentially be utilized to guide stratification of MM patients for immune therapies.

Spatial immunophenotypes predict response to anti-PD1 treatment and capture distinct paths of T cell evasion in triple negative breast cancer.

Only a subgroup of triple-negative breast cancer (TNBC) responds to immune checkpoint inhibitors (ICI). To better understand lack of response to ICI, we analyze 681 TNBCs for spatial immune cell contextures in relation to clinical outcomes and pathways of T cell evasion. Excluded, ignored and inflamed phenotypes can be captured by a gene classifier that predicts prognosis of various cancers as well as anti-PD1 response of metastatic TNBC patients in a phase II trial. The excluded phenotype, which is associated with resistance to anti-PD1, demonstrates deposits of collagen-10, enhanced glycolysis, and activation of TGFß/VEGF pathways; the ignored phenotype, also associated with resistance to anti-PD1, shows either high density of CD163+ myeloid cells or activation of WNT/PPARγ pathways; whereas the inflamed phenotype, which is associated with response to anti-PD1, revealed necrosis, high density of CLEC9A+ dendritic cells, high TCR clonality independent of neo-antigens, and enhanced expression of T cell co-inhibitory receptors.

Differential quantities of immune checkpoint-expressing CD8 T cells in soft tissue sarcoma subtypes.

INTRODUCTION: Local T-cell immunity is recognized for its contribution to the evolution and therapy response of various carcinomas. Here, we investigated characteristics of tumor-infiltrating lymphocytes (TILs), as well as T-cell evasive mechanisms in different soft tissue sarcoma (STS) subtypes. METHODS: Liposarcoma, gastrointestinal stromal tumor (GIST), leiomyosarcoma, myxofibrosarcoma and pleomorphic sarcomas were assessed for T-cell numbers and phenotypes using flow cytometry. Next-generation sequencing was used to analyze T-cell receptor repertoire, mutational load, immune cell frequencies, and expression of immune-related genes. RESULTS: GIST, myxofibrosarcoma and pleomorphic sarcoma showed high numbers of CD8+ TILs, with GIST having the lowest fraction of effector memory T cells. These TILs coexpress the immune checkpoints PD1, TIM3, and LAG3 in myxofibrosarcoma and pleomorphic sarcoma, yet TILs coexpressing these checkpoints were near negligible in GIST. Fractions of dominant T-cell clones among STS subtypes were lowest in GIST and liposarcoma, whereas mutational load was relatively low in all STS subtypes. Furthermore, myeloid-derived cells and expression of the costimulatory ligands CD86, ICOS-L and 41BB-L were lowest in GIST when compared with other STS subtypes. CONCLUSION: STS subtypes differ with respect to number and phenotypical signs of antitumor responsiveness of CD8+ TILs. Notably, GIST, myxofibrosarcoma and pleomorphic sarcoma harbor high numbers of CD8+ T cells, yet in the GIST microenvironment, these T cells are less differentiated and non-exhausted, which is accompanied with a relatively low expression of costimulatory ligands.