Exploring the impact of breast cancer on colonization resistance of mouse microbiota using network node manipulation.

Breast cancer, a global health concern affecting women, has been linked to alterations in the gut microbiota, impacting various aspects of human health. This study investigates the interplay between breast cancer and the gut microbiome, particularly focusing on colonization resistance-an essential feature of the microbiota's ability to prevent pathogenic overgrowth. Using a mouse model of breast cancer, we employ diversity analysis, co-occurrence network analysis, and robustness tests to elucidate the impact of breast cancer on microbiome dynamics. Our results reveal that breast cancer exposure affects the bacterial community's composition and structure, with temporal dynamics playing a role. Network analysis demonstrates that breast cancer disrupts microbial interactions and decreases network complexity, potentially compromising colonization resistance. Moreover, network robustness analysis shows the susceptibility of the microbiota to node removal, indicating potential vulnerability to pathogenic colonization. Additionally, predicted metabolic profiling of the microbiome highlights the significance of the enzyme EC 6.2.1.2 - Butyrate--CoA ligase, potentially increasing butyrate, and balancing the reduction of colonization resistance. The identification of Rubrobacter as a key contributor to this enzyme suggests its role in shaping the microbiota's response to breast cancer. This study uncovers the intricate relationship between breast cancer, the gut microbiome, and colonization resistance, providing insights into potential therapeutic strategies and diagnostic approaches for breast cancer patients.

The antitumor effect induced by an IL-2 'no-alpha' mutein depends on changes in the CD8+ T lymphocyte/Treg cell balance.

High doses of interleukin-2 (IL-2) have been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy, with a ~15% response rate. Remarkably, 7%-9% of patients achieve complete or long-lasting responses. Many patients treated with IL-2 experienced an expansion of regulatory T cells (Tregs), specifically the expansion of ICOS+ highly suppressive Tregs, which correlate with worse clinical outcomes. This partial efficacy together with the high toxicity associated with the therapy has limited the use of IL-2-based therapy. Taking into account the understanding of IL-2 structure, signaling, and in vivo functions, some efforts to improve the cytokine properties are currently under study. In previous work, we described an IL-2 mutein with higher antitumor activity and less toxicity than wtIL-2. Mutein was in silico designed for losing the binding capacity to CD25 and for preferential stimulation of effector cells CD8+ and NK cells but not Tregs. Mutein induces a higher anti-metastatic effect than wtIL-2, but the extent of the in vivo antitumor activity was still unexplored. In this work, it is shown that mutein induces a strong antitumor effect on four primary tumor models, being effective even in those models where wtIL-2 does not work. Furthermore, mutein can change the in vivo balance between Tregs and T CD8+ memory/activated cells toward immune activation, in both healthy and tumor-bearing mice. This change reaches the tumor microenvironment and seems to be the major explanation for mutein efficacy in vivo.

Blocking IL-2 Signal In Vivo with an IL-2 Antagonist Reduces Tumor Growth through the Control of Regulatory T Cells.

IL-2 is critical for peripheral tolerance mediated by regulatory T (Treg) cells, which represent an obstacle for effective cancer immunotherapy. Although IL-2 is important for effector (E) T cell function, it has been hypothesized that therapies blocking IL-2 signals weaken Treg cell activity, promoting immune responses. This hypothesis has been partially tested using anti-IL-2 or anti-IL-2R Abs with antitumor effects that cannot be exclusively attributed to lack of IL-2 signaling in vivo. In this work, we pursued an alternative strategy to block IL-2 signaling in vivo, taking advantage of the trimeric structure of the IL-2R. We designed an IL-2 mutant that conserves the capacity to bind to the αß-chains of the IL-2R but not to the γc-chain, thus having a reduced signaling capacity. We show our IL-2 mutein inhibits IL-2 Treg cell-dependent differentiation and expansion. Moreover, treatment with IL-2 mutein reduces Treg cell numbers and impairs tumor growth in mice. A mathematical model was used to better understand the effect of the mutein on Treg and E T cells, suggesting suitable strategies to improve its design. Our results show that it is enough to transiently inhibit IL-2 signaling to bias E and Treg cell balance in vivo toward immunity.

Deciphering the molecular bases of the biological effects of antibodies against Interleukin-2: a versatile platform for fine epitope mapping.

Elucidating the network of interactions established by Interleukin-2 is a key step to understanding its role as a master regulator of the immune system. Binding of this cytokine by specific antibodies gives rise to different classes of immune complexes that boost or inhibit immune responses. The molecular bases of such functional dichotomy are likely related to the nature of the recognized epitopes, making it necessary to perform fine epitope mapping studies. The current work was aimed at developing a versatile platform to do so. This was accomplished by display of human and mouse Interleukin-2 on filamentous phages, together with extensive mutagenesis of both antigens and high throughput screening of binding properties of more than 200 variants. Detailed molecular pictures of the epitopes were thus delineated for four antibodies against either human or mouse Interleukin-2, which refined and, in some cases, modified the conclusions derived from previous mapping studies with peptide libraries. Overlapping surface patches on mouse Interleukin-2 that also coincide with the predicted interface between the cytokine and its receptor alpha chain were shown to be recognized by two monoclonal antibodies that promote enhancement of immune responses, shedding new light on the structural bases of their biological activity. Our strategy was powerful enough to reveal multiple binding details and could be used to map the epitopes recognized by other antibodies and to explore additional interactions involving Interleukin-2 and related cytokines, thus contributing to our understanding of the complex structure-function relationships within the immune system.

Chemotherapy induced transient B-cell depletion boosts antibody-forming cells expansion driven by an epidermal growth factor-based cancer vaccine.

Cancer vaccines efficacy may improve inducing a rapid and persistent immune response, early at diagnosis along with standard therapies. EGF chemically conjugated to the carrier protein P64k from Neisseria meningitidis in montanide ISA 51 adjuvant is under evaluation, aiming to stimulate a B-cell response. High-dose cyclophosphamide and doxorubicin after priming enhanced the long-term frequency of EGF-specific antibody-forming cells (AFC) of IgM and IgG isotypes, but not the P64k response. Resulting combination, limitedly operational in Btk deficient xid mice, suggests that preferential B-cell lymphocyte space promoted by cyclophosphamide facilitates remaining EGF-specific AFC undergo homeostatic proliferation driven by boosting, amplifying the response.

Priming and boosting determinants on the antibody response to an Epidermal Growth Factor-based cancer vaccine.

Epidermal Growth Factor chemically conjugated to P64k carrier protein from Neisseria meningitidis emulsified in Montanide ISA 51 adjuvant is a cancer vaccine under clinical evaluation. We explored the influence of priming and boosting variables on the antibody response in mice. An apparently low dose fractionated in multiple anatomical sites at priming accelerated the induction and enhanced the maximal antibody response, with a long-lasting effect. Moreover, shortening the boosting time reduces the antibody persistence. Repeatedly boosting shift subjects to good antibody-responders, maintaining the epitope immunodominance. We conclude that optimizing immunopharmacological determinants contribute to an earlier, stronger and prolonged anti-EGF antibody persistence.