A Technical Framework for Investigating Microbial Antigen-Driven Spatial Immune Selection and Clonal Escape in Acquired Aplastic Anemia

Abstract

Acquired aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome in which hematopoietic stem and progenitor cell (HSPC) destruction coexists with selective expansion of hematopoietic clones capable of surviving immune pressure. Recent studies have independently demonstrated virus-reactive T-cell receptors (TCRs) with crossreactivity against hematopoietic progenitor-cell antigens, disease-associated TCR signatures, spatially organized inflammatory marrow microenvironments, and recurrent somatic mechanisms of immune escape involving human leukocyte antigen (HLA) loss and other clonal alterations. However, these observations remain largely disconnected, and no operational framework has established how a candidate microbial antigen should be evaluated across the successive stages of temporal exposure, HLA-restricted immune recognition, spatial HSPC injury, and immune-selected clonal escape. This technical report proposes a prospective, hypothesis-generating microbe-immune-niche-clone framework in which the individual patient constitutes the primary unit of mechanistic inference. Its principal novelty lies not in proposing infection as a new association with AA, but in defining a prespecified and falsifiable evidentiary pathway that separates microbial detection from microbial causation. The framework integrates pretreatment and longitudinal sampling, conventional marrow pathology, spatial immune characterization, high-resolution HLA analysis, paired blood and marrow TCR repertoire profiling, sensitive paroxysmal nocturnal hemoglobinuria testing, somatic genomic analysis, clinically directed microbiological testing, pathogen-agnostic metagenomic sequencing where appropriate, computational antigen matching, and functional validation of candidate HLA-TCR-antigen relationships. A six-level evidence hierarchy, extending from absence of a microbial signal through temporal, immunogenetic, spatial-clonal, and functional concordance, is accompanied by explicit negative, non-evaluable, and falsifying pathways to reduce confirmation bias and retrospective causal attribution. Advanced microbial, spatial, single-cell, and functional assays are investigational and are not proposed as components of routine AA diagnostic evaluation. The framework is intended to identify mechanistically coherent individual cases and, if reproducible across independent patients, candidate biological subgroups; it is not designed by itself to establish population-level microbial causality or to alter established diagnostic, antimicrobial, immunosuppressive, or transplantation pathways