Tumor-reactive T cell clonotype dynamics underlying clinical response to TIL therapy in melanoma.

Adoptive cell therapy (ACT) using in vitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and T cell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon in vitro expansion, yielding products enriched in tumor-specific CD8+ cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization.

Cryoablation Does Not Significantly Contribute to Systemic Effector Immune Responses in a Poorly Immunogenic B16F10 Melanoma Model.

PURPOSE: Cryoablation is a minimally invasive procedure implemented to destroy solid tumors. It also results in the release of tumor antigens into the systemic circulation. Preclinical studies using immunogenic tumor models have shown that cryoablation evokes antitumor immune responses. The mechanisms by which cryoablation impacts immune responses in poorly immunogenic tumors have not been sufficiently explored. EXPERIMENTAL DESIGN: We used a bilateral B16F10 melanoma model devoid of strong immunogenic antigens. Cryoablation-induced effector immune responses were investigated, also in combination with a peritumoral STING agonist and systemic anti-PD-1. Selective immune cell depletion, T-cell migration arrest, in vivo T-cell transplantation, and cryoablation versus surgical removal techniques were used to determine the contribution of cryoablation and immunotherapies to systemic antitumor effector immune responses. RESULTS: Treatment of a tumor with cryoablation + STING agonist + anti-PD-1 resulted in the rejection of unablated, contralateral tumors. Depletion studies demonstrated that tumor rejection is essentially dependent on CD8+ T cells. T-cell arrest in the lymph nodes had no effect on the rejection process. Splenic CD8+ T cells isolated from cryoablation-treated mice with B16F10 melanoma, upon transplantation into melanoma-bearing recipients, did not impact the recipient's tumor growth. Finally, comparison of cryoablation + STING agonist + anti-PD-1 versus surgery + STING agonist + anti-PD-1 in the bilateral tumor model showed no difference in the rejection of contralateral tumors. CONCLUSIONS: Cryoablation does not significantly contribute to systemic antitumor effector immune responses in a B16F10 melanoma model. Cryoablation primarily performs tumor debulking, and immunotherapy functions independently of cryoablation in eliciting antitumor effector immune responses.

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma.

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8+ T cells.

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.

Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy.

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell-infiltrated tumors. SIGNIFICANCE: Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1.

Interpretation of T cell states from single-cell transcriptomics data using reference atlases.

Single-cell RNA sequencing (scRNA-seq) has revealed an unprecedented degree of immune cell diversity. However, consistent definition of cell subtypes and cell states across studies and diseases remains a major challenge. Here we generate reference T cell atlases for cancer and viral infection by multi-study integration, and develop ProjecTILs, an algorithm for reference atlas projection. In contrast to other methods, ProjecTILs allows not only accurate embedding of new scRNA-seq data into a reference without altering its structure, but also characterizing previously unknown cell states that "deviate" from the reference. ProjecTILs accurately predicts the effects of cell perturbations and identifies gene programs that are altered in different conditions and tissues. A meta-analysis of tumor-infiltrating T cells from several cohorts reveals a strong conservation of T cell subtypes between human and mouse, providing a consistent basis to describe T cell heterogeneity across studies, diseases, and species.

Modulation of CD4 T cell function via CD6-targeting.

In recent years molecules involved on the immune synapse became successful targets for therapeutic immune modulation. CD6 has been extensively studied, yet, results regarding CD6 biology have been controversial, in spite of the ubiquitous presence of this molecule on virtually all CD4 T cells. We investigated the outcome of murine and human antibodies targeting CD6 domain 1. We found that CD6-targeting had a major impact on the functional specialization of CD4 cells, both human and murine. Differentiation of CD4 T cells towards a Foxp3+ Treg fate was prevented with increasing doses of anti-CD6, while Th1 polarization was favoured. No impact was observed on Th2 or Th17 specialization. These in vitro results provided an explanation for the dose-dependent outcome of in vivo anti-CD6 administration where the anti-inflammatory action is lost at the highest doses. Our data show that therapeutic targeting of the immune synapse may lead to paradoxical dose-dependent effects due to modification of T cell fate.

Evidencias de la regulación del estado redox materno entre las 30 y 36 semanas de gestación / Evidences of the maternal redox state regulation among the 30 and the 36 weeks of pregnancy

El embarazo es un estado fisiológico caracterizado por un incremento regulado estrictamente de los procesos oxidativos. El riesgo de estrés oxidativo puede depender del estado antioxidante materno, el cual protege potencialmente a la unidad materno-fetal incrementando el crecimiento intrauterino y el peso del neonato. OBJETIVO: Mostrar evidencias de la regulación del estado redox materno entre las 30 y 36 sem de gestación fisiológica. MÉTODOS: Se realizó un estudio observacional, analítico y transversal que incluyó a 36 gestantes saludables con feto único, similar edad, peso y edades gestacionales entre 30 y 36 sem. Como indicadores de defensa antioxidante se determinaron el potencial reductor férrico del suero, la actividad de la superóxido dismutasa extracelular y la concentración de glutatión reducido en eritrocitos. Como indicador de lipoperoxidación se determinó la concentración de malondialdehído. Se determinó además la concentración de hemoglobina, hierro sérico y la glicemia, así como, el índice de masa corporal (IMC) y su variación. Basados en la metodología de análisis de senderos, se contrastaron modelos de ecuaciones estructurales con las variables estudiadas. RESULTADOS: El potencial reductor férrico y la concentración sérica de malondialdehído correlacionaron positivamente con la actividad sérica de la superóxido dismutasa y con la concentración de glutatión reducido, contribuyendo más el malondialdehído que el potencial reductor férrico a su variación. La glicemia también se relacionó directamente con la peroxidación lipódica y con los indicadores del estado antioxidante. CONCLUSIONES: Las gestantes incluidas en el estudio tuvieron un adecuado balance redox y el malondialdehído o algún otro producto de la peroxidación lipídica pueden estar relacionados con la estimulación de respuestas protectoras para mantener el mismo (AU)

Pregnancy is a physiological status characterized by a strictly controlled increase of oxidative processes. The risk of this oxidative process may to depend on the mother antioxidant status, which to potentially protect the mother-fetus unit increasing the intrauterine growth and the neonate weight. OBJECTIVE: To show evidences of mother redox satus regulation among the 30 and the 36 weeks of physiological pregnancy. METHODS: A cross-sectional, analytical and observational study was conducted including 36 healthy pregnants wit a single-fetus, and similar weight and gestational age between 30 and 36 weeks. As antioxidant defense indicators were determined the potential serum-ferric reduction agent, the extracellular superoxide dismutase and malondialdehyde concentration. It was also determined the hemoglobin ,serum ferric and glycemia, as well as the body mass index (BMI) and its variation. Based on the methodology of routes analysis there were structural equations forms with study variable. RESULTS: The ferric redactor potential and the serum concentration of malondialdehye were positively correlated to serum activity of superoxide dismutase and to the reduced glutathione concentration were the malondialdehyde contributes more than the ferric redactor potential and its variation. Glycemia also was directly related to lipid peroxidation and to antioxidant status indicators. CONCLUSIONS: Pregnants included in present study had a suitable redox balance, and the malondialdehyde or some other product of lipid peroxidation may be related to stimulation of protective responses for its maintenance (AU)

Evidencias de la regulación del estado redox materno entre las 30 y 36 semanas de gestación / Evidences of the maternal redox state regulation among the 30 and the 36 weeks of pregnancy

El embarazo es un estado fisiológico caracterizado por un incremento regulado estrictamente de los procesos oxidativos. El riesgo de estrés oxidativo puede depender del estado antioxidante materno, el cual protege potencialmente a la unidad materno-fetal incrementando el crecimiento intrauterino y el peso del neonato. OBJETIVO: Mostrar evidencias de la regulación del estado redox materno entre las 30 y 36 sem de gestación fisiológica. MÉTODOS: Se realizó un estudio observacional, analítico y transversal que incluyó a 36 gestantes saludables con feto único, similar edad, peso y edades gestacionales entre 30 y 36 sem. Como indicadores de defensa antioxidante se determinaron el potencial reductor férrico del suero, la actividad de la superóxido dismutasa extracelular y la concentración de glutatión reducido en eritrocitos. Como indicador de lipoperoxidación se determinó la concentración de malondialdehído. Se determinó además la concentración de hemoglobina, hierro sérico y la glicemia, así como, el índice de masa corporal (IMC) y su variación. Basados en la metodología de análisis de senderos, se contrastaron modelos de ecuaciones estructurales con las variables estudiadas. RESULTADOS: El potencial reductor férrico y la concentración sérica de malondialdehído correlacionaron positivamente con la actividad sérica de la superóxido dismutasa y con la concentración de glutatión reducido, contribuyendo más el malondialdehído que el potencial reductor férrico a su variación. La glicemia también se relacionó directamente con la peroxidación lipódica y con los indicadores del estado antioxidante. CONCLUSIONES: Las gestantes incluidas en el estudio tuvieron un adecuado balance redox y el malondialdehído o algún otro producto de la peroxidación lipídica pueden estar relacionados con la estimulación de respuestas protectoras para mantener el mismo

Pregnancy is a physiological status characterized by a strictly controlled increase of oxidative processes. The risk of this oxidative process may to depend on the mother antioxidant status, which to potentially protect the mother-fetus unit increasing the intrauterine growth and the neonate weight. OBJECTIVE: To show evidences of mother redox satus regulation among the 30 and the 36 weeks of physiological pregnancy. METHODS: A cross-sectional, analytical and observational study was conducted including 36 healthy pregnants wit a single-fetus, and similar weight and gestational age between 30 and 36 weeks. As antioxidant defense indicators were determined the potential serum-ferric reduction agent, the extracellular superoxide dismutase and malondialdehyde concentration. It was also determined the hemoglobin ,serum ferric and glycemia, as well as the body mass index (BMI) and its variation. Based on the methodology of routes analysis there were structural equations forms with study variable. RESULTS: The ferric redactor potential and the serum concentration of malondialdehye were positively correlated to serum activity of superoxide dismutase and to the reduced glutathione concentration were the malondialdehyde contributes more than the ferric redactor potential and its variation. Glycemia also was directly related to lipid peroxidation and to antioxidant status indicators. CONCLUSIONS: Pregnants included in present study had a suitable redox balance, and the malondialdehyde or some other product of lipid peroxidation may be related to stimulation of protective responses for its maintenance

Balance entre las especies reactivas y los sistemas antiooxidantes en la gestación normal / Balance between reactive species and antioxidant systems in a normal pregnancy

La gestación es un estado caracterizado por un incremento, estrictamente regulado, de los procesos oxidativos en la madre y en el producto de la concepción, determinado por un aumento en el consumo de dioxígeno, y por la utilización de algunas especies reactivas en varios procesos celulares importantes para el desarrollo materno-fetal. El desbalance a corto y/o largo plazo del equilibrio antioxidantes/prooxidantes a favor de estos últimos provoca estrés oxidativo y daño a biomoléculas, pérdida de sus funciones y muerte celular, lo que pudiera afectar la evolución normal de la gestación. Por este motivo resulta importante conocer cómo se regula el estado redox en este período. En la presente revisión se muestran algunos de los principales estudios y se discuten resultados que evidencian el papel de las especies reactivas y los sistemas antioxidantes en la gestación normal.(AU)

Pregnancy is a state characterizing by an increase, strictly regulated of oxidative processes in mother and in fetus, determined by an increase of dioxygen consumption, and by use of some reactive species in some cellular processes significant for mother-fetus development. Short- and long-term lack of anti-oxidant/pro-oxidant balance, favouring these later, provoke oxidative stress and damage to bio-molecules, loss of functions and apoptosis, affecting the normal course of pregnancy. Thus, it is important to know regulation of redox state in this period. In present review are showed some of main studies and results discussed evidencing role of Reactive Species and Anti-oxidant systems in normal pregnancy(AU)

Balance entre las especies reactivas y los sistemas antiooxidantes en la gestación normal / Balance between reactive species and antioxidant systems in a normal pregnancy

La gestación es un estado caracterizado por un incremento, estrictamente regulado, de los procesos oxidativos en la madre y en el producto de la concepción, determinado por un aumento en el consumo de dioxígeno, y por la utilización de algunas especies reactivas en varios procesos celulares importantes para el desarrollo materno-fetal. El desbalance a corto y/o largo plazo del equilibrio antioxidantes/prooxidantes a favor de estos últimos provoca estrés oxidativo y daño a biomoléculas, pérdida de sus funciones y muerte celular, lo que pudiera afectar la evolución normal de la gestación. Por este motivo resulta importante conocer cómo se regula el estado redox en este período. En la presente revisión se muestran algunos de los principales estudios y se discuten resultados que evidencian el papel de las especies reactivas y los sistemas antioxidantes en la gestación normal.

Pregnancy is a state characterizing by an increase, strictly regulated of oxidative processes in mother and in fetus, determined by an increase of dioxygen consumption, and by use of some reactive species in some cellular processes significant for mother-fetus development. Short- and long-term lack of anti-oxidant/pro-oxidant balance, favouring these later, provoke oxidative stress and damage to bio-molecules, loss of functions and apoptosis, affecting the normal course of pregnancy. Thus, it is important to know regulation of redox state in this period. In present review are showed some of main studies and results discussed evidencing role of Reactive Species and Anti-oxidant systems in normal pregnancy.