T-cells produce acidic niches in lymph nodes to suppress their own effector functions

The immune system is a complex network of cells and organs working together to defend the body against pathogens and other harmful substances. Lymph nodes (LNs) are crucial secondary lymphoid organs that play a central role in adaptive immunity. They serve as sites where immune cells, including T and B lymphocytes, interact with antigen-presenting cells (APCs), initiating and regulating immune responses. The LN microenvironment is complex and involves a precise interplay between various cell types and soluble factors within distinct anatomical zones. While the histological organization and cellular interactions within LNs are relatively well understood, the precise physiological microenvironment and its influence on immune cell function remain less characterized. Previous research has established that acidic pH in the tumor microenvironment significantly inhibits the effector functions of activated CD8+ T cells. This acidic environment limits T-cell proliferation, cytokine production, and cytotoxic activity, contributing to immune evasion and tumor progression. This study explores the possibility that this acidic microenvironment isn't solely a pathological phenomenon in the context of cancer but also plays a physiological role in immune regulation within the healthy immune system, specifically within LNs. The researchers investigate the hypothesis that the unique microenvironment of lymph nodes, characterized by low convective flow and high glucose metabolism, naturally promotes an acidic pH. This low pH, similar to the tumor microenvironment, is predicted to act as a negative feedback mechanism controlling the activation and effector functions of T cells. The importance of understanding this potential physiological role of acidity is based on the need to improve therapeutic strategies in cancer treatment and to gain a more comprehensive understanding of the complexities of immune regulation.

The literature review extensively examines the impact of extracellular pH on immune cell function, focusing particularly on T cells. Studies have demonstrated the suppressive effects of acidic conditions on T-cell proliferation, cytokine production, and cytotoxicity. This inhibition is linked to the disruption of cellular metabolism, specifically glycolysis, through the impact of pH on ion transporters like monocarboxylate transporters (MCTs). The researchers refer to several studies showing the crucial role of T-cell metabolism in their effector functions, emphasizing the tight interplay between metabolic pathways and immune responses. Moreover, the literature review highlights the known acidosis in the tumor microenvironment and its contribution to immune evasion, which provides the rationale for investigating the possibility of a parallel, physiological role of acidity in immune regulation within healthy lymphoid tissues like lymph nodes. Several studies are referenced showing the impact of acidity on both innate and adaptive immune cells. The cited works highlight the significant influence of the extracellular environment on immune cell function, emphasizing the need to further investigate the physiological roles of these environmental factors.

The study utilized a multi-faceted approach combining in vivo imaging techniques, pharmacological interventions, and in vitro cellular assays. In vivo fluorescence and magnetic resonance (MR) imaging were employed to assess the pH levels within lymph nodes of mice. A pH-sensitive probe, pHLIp (pH low insertion peptide), was used for fluorescence imaging. This probe is designed to accumulate preferentially in acidic regions, enabling visualization of acidic niches within the lymph node architecture. The use of MR imaging, specifically chemical exchange saturation transfer (CEST) MRI, provided a non-invasive method to confirm the pH measurements obtained with the pHLIp probe. To investigate the role of T cells in creating the acidic microenvironment, experiments were performed on athymic nude mice (lacking T cells) and on mice subjected to acute lymphopenia induced by the administration of anti-CD4 and anti-CD8 antibodies. This allowed the researchers to assess the relationship between T-cell presence and lymph node acidity. To determine if carbonic anhydrases were involved in pH regulation, mice were treated with inhibitors of these enzymes prior to imaging. To explore the effect of pH on T-cell glycolysis, the researchers measured extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) in T cells cultured at different pH levels using a Seahorse XF analyzer. In vitro experiments were designed to measure glucose consumption, lactate production, and the expression of MCTs under varying pH conditions. The impact of pH on T-cell activation and cytokine production was assessed using in vitro assays measuring IFN-γ and IL-2 secretion by stimulated T cells. Flow cytometry was also employed to analyze T-cell activation markers, along with intracellular cytokine staining. To investigate additional mechanisms for pH-sensing, various acid-sensing receptors and ion channels were examined in knockout mice and/or using pharmacological inhibitors, measuring cytokine release in response to varying pH levels. Statistical analyses, including ANOVA and t-tests, were performed to compare the results between experimental groups.

The study's key findings demonstrate the existence of acidic niches within the paracortical zones of lymph nodes in vivo. These acidic regions are found to be substantially lower in pH than surrounding tissues, averaging around 6.3. Crucially, the formation of these acidic microenvironments is directly linked to the presence of T cells, as indicated by the absence of acidic niches in athymic nude mice and in mice with experimentally induced lymphopenia. The researchers linked this acidic pH to the suppression of T-cell effector functions. In vitro experiments showed that low pH significantly inhibits T-cell glycolysis through the acid-mediated inhibition of monocarboxylate transporters (MCTs). This leads to a negative feedback loop, where acid production by glycolysis is reduced in response to low pH. Interestingly, despite this suppression of glycolysis and cytokine production in vitro, the initial activation of naive T cells by dendritic cells was not found to be hindered by low pH, suggesting a potential role for the acidic environment in regulating T-cell activation. The investigation of alternative mechanisms of acidity regulation ruled out a major role of carbonic anhydrases. The in vivo findings were validated using CEST MRI, independently confirming the acidic nature of the lymph node microenvironment. The overall findings suggest a potential mechanism by which the immune system creates acidic niches in LNs to regulate T-cell function, primarily by suppressing cytokine production and promoting a feedback loop to control glycolytic activity. Specific inhibition of IFNγ cytokine release under acidic conditions was demonstrated, while the impact on other cytokines was more varied. In contrast, T-cell activation itself was not affected by the acidic environment.

The findings of this study challenge the conventional understanding of the lymph node microenvironment, highlighting the physiological significance of acidity in immune regulation. The researchers demonstrate that T cells themselves actively contribute to generating an acidic environment within lymph nodes. This acidic microenvironment acts as a regulatory mechanism to control T-cell activation and cytokine release, potentially preventing excessive inflammation and tissue damage. The observation that T-cell activation itself is unaffected by this acidic environment is crucial, suggesting that the acidic niche may play a specific role in the regulation of effector T-cell functions rather than in the initial activation process. This precise control of T-cell activity within the lymph node microenvironment has important implications for immune homeostasis and potentially for the development of immune-related diseases and therapeutic strategies in cancer immunotherapy. The data demonstrates a novel negative feedback mechanism regulating T cell activity via pH, adding another layer of complexity to the dynamics of immune responses. Future research should focus on exploring the potential clinical implications of this finding, particularly in relation to immunotherapies that aim to enhance T-cell responses against tumors.

This study demonstrates for the first time that T cells generate acidic niches within lymph nodes, which act as a physiological mechanism to suppress their own effector functions. The acidity primarily affects cytokine release without impairing T-cell activation. This discovery highlights the complex interplay between cellular metabolism and immune regulation and offers new perspectives on the development of immune-modulatory therapies. Future research should focus on exploring the precise molecular mechanisms involved in the pH-dependent regulation of T-cell functions and on investigating the potential for manipulating this mechanism to enhance or suppress immune responses in various disease contexts.

While the study provides compelling evidence for the role of T-cell-mediated acidity in regulating effector functions, several limitations need consideration. The study is primarily based on murine models, and the extent to which these findings translate directly to human physiology remains to be determined. The use of pH-sensitive probes and imaging techniques might be subject to limitations in terms of spatial and temporal resolution. Furthermore, the mechanisms beyond MCT inhibition that might contribute to pH-dependent T-cell suppression should be more thoroughly investigated. Lastly, the precise roles of various cytokines under acidic conditions should be explored more extensively to provide better insights into the complexity of this regulatory mechanism.