The increasing frequency of general and particularly male cancer coupled with the reduction in male fertility seen worldwide motivated us to seek a potential evolutionary link between these two phenomena, concerning the reproductive transcriptional modules observed in cancer and the expression of cancer-testis antigens (CTA). The phylostratigraphy analysis of the human genome allowed us to link the early evolutionary origin of cancer via the reproductive life cycles of the unicellulars and early multicellulars, potentially driving soma-germ transition, female meiosis, and the parthenogenesis of polyploid giant cancer cells (PGCCs), with the expansion of the CTA multi-families, very late during their evolution. CTA adaptation was aided by retrovirus domestication in the unstable genomes of mammals, for protecting male fertility in stress conditions, particularly that of humans, as compensation for the energy consumption of a large complex brain which also exploited retrotransposition. We found that the early and late evolutionary branches of human cancer are united by the immunity-proto-placental network, which evolved in the Cambrian and shares stress regulators with the finely-tuned sex determination system. We further propose that social stress and endocrine disruption caused by environmental pollution with organic materials, which alter sex determination in male foetuses and further spermatogenesis in adults, bias the development of PGCC-parthenogenetic cancer by default.

IgA nephropathy (IgAN) is characterised by the production of galactose-deficient IgA1 (GdIgA1) antibodies. As the source of pathogenic antibodies, B cells are central to IgAN pathogenesis, but the B cell activation pathways as well as the potential B cell source of dysregulated IgA-secretion remain unknown. We carried out flow cytometry analysis of peripheral blood B cells in patients with IgA nephropathy and control subjects with a focus on IgA-expressing B cells to uncover the pathways of B cell activation in IgAN and how these could give rise to pathogenic GdIgA1 antibodies. In addition to global changes in the B cell landscape – expansion of naive and reduction in memory B cells – IgAN patients present with an increased frequency of IgA-expressing B cells that lack the classical memory marker CD27, but are CD21^pos. IgAN patients further have an expanded population of IgA^pos antibody-secreting cells, which correlate with serum IgA levels. Both IgA^pos plasmabalsts and CD27^neg B cells co-express GdIgA1. Implicating dysregulation at mucosal surfaces as the driver of such B cell differentiation, we found a correlation between lipopolysaccharide (LPS) in the serum and IgA^posCD27^neg B cell frequency. We propose that dysregulated immunity in the mucosa may drive de novo B cell activation within germinal centres, giving rise to IgA^posCD27^neg B cells and subsequently IgA-producing plasmablasts. These data integrate B cells into the paradigm of IgAN pathogenesis and allow to further investigate this pathway to uncover biomarkers and develop therapeutic interventions.

The increasing frequency of male cancer coupled with the reduction in male fertility seen worldwide motivated us to seek a potential evolutionary link between these two phenomena, concerning the reproductive transcriptional modules observed in cancer and the expression of cancer-testis-antigens (CTA). The phylostratigraphy analysis of the human genome allowed us to link the early evolutionary origin of cancer by reproductive life cycles of the unicellulars and early multicellulars, potentially driving soma-germ transition, female meiosis and parthenogenesis of polyploid giant cancer cells (PGCCs), with the expansion of the CTA multi-families, very late during evolution. CTA adaptation was aided by retrovirus domestication in the unstable genomes of mammals, for protecting male fertility in stress conditions, particularly that of humans, as compensation for the energy consumption by a large complex brain which also exploited retrotransposition. We found that the early and late evolutionary branches of human cancer are united by the immunity-proto-placental network, which evolved in the Cambrian and shares stress regulators with the finely-tuned sex determination system. We further propose that social stress and endocrine disruption caused by environmental pollution with organic materials, which alter sex determination in male foetuses and further spermatogenesis in adults, bias the development of PGCC-parthenogenetic cancer by default.

Chronic lymphocytic leukemia (CLL) is associated with immune dysfunction, but how disease-intrinsic mechanisms in treatment-naive patients influence the coordination of adaptive responses to novel antigens remains unclear. Here, we assessed SARS-CoV-2-specific antibody and T cell immunity in 38 treatment-naive CLL patients and 13 healthy controls (HCs) following vaccination. Despite significantly reduced total immunoglobulin levels compared to HCs, 94.7% of CLL patients developed SARS-CoV-2-specific IgG, and 89.5% mounted IgA responses, with serum titers comparable to those of HCs. Virus-specific T cell responses, measured by IFN-γ release following antigen stimulation, were detected in 78.9% of patients. CLL patients had significantly more circulating CD4⁺ T follicular helper (Tfh) and T follicular regulatory (Tfr) cells than HCs. These expansions correlated with B cell abundance, which, in untreated CLL, predominantly reflects malignant B cells. Notably, Tfh cell frequencies and absolute counts were highest in patients lacking a SARS-CoV-2-specific T cell response, indicating a decoupling between Tfh expansion and functional antiviral immunity. Overall, these findings demonstrate that while SARS-CoV-2-specific immune responses are largely preserved in treatment-naive CLL patients, disease-driven alterations in T cell composition may compromise the coordination and quality of antigen-specific T cell-mediated immunity.

Current gene transfer methods often lack the precision, versatility, or efficiency when integrating large transgenes, limiting the ability to engineer therapeutic T-cells with more complex payloads. Here, we report ‘one-pot’ PASTA (Programmable and Site-specific Transgene Addition), a non-viral genome engineering strategy for large gene insertion that combines CRISPR-Cas-mediated homology-directed repair (HDR) and site-specific recombination via serine integrases. Using ‘one-pot’ PASTA with the Bxb1 integrase, we demonstrate efficient integration of transgenes at multiple genomic loci relevant for T-cell engineering (e.g., TRAC, B2M, CD3E, CD3Z, GAPDH ). For constructs > 8 kb, ‘one-pot’ PASTA outperforms conventional HDR by 19-fold on average and prime-editing-assisted site-specific integrase gene editing (PASSIGE) by 5-fold. This enables the delivery of multi-cistronic cargo to generate dual-antigen targeting CAR T-cells with a safety-switch that overcome antigen escape in lymphoma models. Finally, ‘one-pot’ PASTA can be further optimized with improved integrase enzymes, such as engineered variants of Pa01 or Bxb1, and plasmids with minimized backbones. In summary, ‘one-pot’ PASTA represents a versatile and scalable platform for precise, non-viral gene insertion in T-cells.

Background: IgA nephropathy (IgAN) is characterised by the production of galactose-deficient IgA1 (Gd-IgA1) antibodies. As the source of pathogenic antibodies, B cells are central to IgAN pathogenesis, but the B cell activation pathways as well as the potential B cell source of dysregulated IgA-secretion remain unknown. Methods: We carried out flow cytometry analysis of peripheral blood B cells in patients with IgA nephropathy and control subjects with a focus on IgA-expressing B cells to uncover the pathways of B cell activation in IgAN and how these could give rise to pathogenic GdIgA1 antibodies. Results: In addition to global changes in the B cell landscape – expansion of naive and reduction in memory B cells – IgAN patients present with an increased frequency of IgA-expressing B cells that lack the classical memory marker CD27, but are CD21 ^pos . IgAN patients further have an expanded population of IgA ^pos antibody-secreting cells, which correlate with serum IgA levels. Both IgA ^pos plasmabalsts and CD27 ^neg B cells co-express GdIgA1. Implicating dysregulation at mucosal surfaces as the driver of such B cell differentiation, we found a correlation between lipopolysaccharide (LPS) in the serum and IgA ^pos CD27 ^neg B cell frequency. Conclusion: We propose that dysregulated immunity in the mucosa may drive de novo B cell activation within germinal centres, giving rise to IgA ^pos CD27 ^neg B cells and subsequently IgA-producing plasmablasts. These data integrate B cells into the paradigm of IgAN pathogenesis and allow to further investigate this pathway to uncover biomarkers and develop therapeutic interventions. Key learning points: What was known: Patients with IgA nephropathy (IgAN) have aberrant production of galactose-deficient IgA1 (Gd-IgA1) and antibodies against it, which together form immune complexes that are deposited in the renal mesangium and lead to kidney damage; this is known as the multi-hit model of IgAN pathogenesis. The multi-hit model centrally implicates B cells as they produce both Gd-IgA1 and antibodies against it, yet B cell activation pathways that lead to aberrant antibody production are absent from the model. Only isolated reports exist describing specific features of B cells that are altered in patients with IgAN, including a reduction in regulatory B cells, increase in toll-like receptor 7 expression in total peripheral blood B cells and elevated frequency of circulating CCR9 ⁺ IgA ⁺ B cells. This study adds: In addition to changes in the overall circulating B cell landscape, differentiation of IgA ⁺ plasmablasts is enhanced in patients with IgAN and their levels correlate with serum IgA. IgA-expressing plasmablast frequency correlates with that of IgA ⁺ CD21 ⁺ B cells, that lack the classical memory B cell marker CD27. Both IgA ⁺ plasmablasts and IgA-expressing CD27 ^- B cells co-express GdIgA1 receptors. IgA ⁺ CD27 ^- CD21 ⁺ B cell frequency correlates with serum lipopolysaccharide (LPS) levels, implicating mucosa in their activation. Potential impact: We uncover the previously unknown B cell activation pathway that appears to be associated with pathogenic IgA secretion in IgAN and integrate this into the multi-hit model of IgAN pathogenesis. This pathway holds potential for further investigation to identify biomarkers and therapeutic targets in IgAN.