Germinal Centers Function as Macromolecular Sanctuaries that Protect Follicular T Cells from Immunotoxin-Mediated Depletion

Abstract Targeted lymphodepletion with CD3 immunotoxins (CD3-ITs) is promising for transplantation and T-cell malignancies, but incomplete tissue depletion limits efficacy. We used a murine anti-CD3 immunotoxin model, intravascular staining, histology, and CD4-iDTR mice to define factors associated with survival of follicular T cells. CD3-IT caused profound systemic T-cell depletion and enrichment of follicular helper (T FH ) and follicular regulatory (T FR ) T cells among surviving CD4 + cells in spleen, Peyer’s patches, and lymph nodes. Ex vivo, CXCR5 + T cells bound CD3-IT comparably to CXCR5 - cells and showed similar susceptibility to CD3-IT-mediated cytotoxicity (IC 50 , 0.471–0.569 nM). In contrast, within the splenic parenchyma, CXCR5 + T cells bound CD3-IT at approximately half the level of CXCR5 − counterparts ( p < 0.05). Histologically, CD3-IT depleted 83% of T cells in splenic T-cell zones, whereas GC T cells were relatively spared. Fc-silent anti-CD40L pretreatment disrupted GC architecture and was associated with a marked reduction in the phenotypic T FH compartment after CD3-IT treatment. In CD4-iDTR mice, free diphtheria toxin (DT) and CD3-IT produced substantially less depletion of GC than T-cell-zone T cells, whereas Fc-silent anti-CD3 mAb down-modulated CD3 on GC-T FH cells. BrdU incorporation did not indicate disproportionate GC-T FH proliferation, and relative sparing with both a 58-kDa free DT and a 278-kDa CD3-IT indicates that size alone is insufficient to explain the effect in intact germinal centers in vivo . Together, these data show that intact GC architecture creates a functional barrier to toxin-mediated depletion of resident follicular T cells.