Alternative pathways for the development of lymphoid structures in humans

Abstract

Lymphoid tissue inducer (LTi) cells are critical for inducing the differentiation of most secondary lymphoid organs (SLOs) in mice. In humans, JAK3 and γc deficiencies result in severe combined immunodeficiency (SCIDs) characterized by an absence of T cells, natural killer cells, innate lymphoid cells (ILCs), and presumably LTi cells. Some of these patients have undergone allogeneic stem cell transplantation (HSCT) in the absence of myeloablation, which leads to donor T cell engraftment, while other leukocyte subsets are of host origin. By using MRI to look for SLOs in nine of these patients 16 to 44 y after HSCT, we discovered that SLOs were exclusively found in the three areas of the abdomen that drain the intestinal tract. A postmortem examination of a child with γc-SCID who had died 3.5 mo after HSCT showed corticomedullary differentiation in the thymus, T cell zones in the spleen, and the appendix, but in neither lymph nodes nor Peyer patches. Tertiary lymphoid organs were observed in the lung. No RAR-related orphan receptor-positive LTi cells could be detected in the existing lymphoid structures. These results suggest that while LTi cells are required for the genesis of most SLOs in humans, SLO in the appendix and in gut-draining areas, as well as tertiary lymphoid organs, can be generated likely by LTi cell-independent mechanisms.

Trial registration: ClinicalTrials.gov NCT04246840.

Keywords: immune system; lymphoid organs; lymphoid tissue.

Fig. 1.

MRI shows that transplanted SCID patients have few lymph nodes. Merged T2-weighted and DWI datasets. In a 25-y-old female patient (P7, Left), no lymph-node-like structures were visible in the thorax (A) or the abdomen (C). In a 31 y-old control (Right), several lymph node structures are visible in the hilar, mediastinal (B), intraperitoneal, and retroperitoneal areas (D) (arrowheads).

Fig. 2.

MRI detects lymph nodes in some areas of the abdomen in transplanted SCID patients. Fusion imaging of T2-weighted sequences and DWI in male patient (P6) (A and C) and controls (B and D). Some large isolated lymph formations are visualized in the retroperitoneal region and in the celio-mesenteric areas in a 27-y-old patient (P6) and a 44-y-old (C, arrowheads) (P1), respectively. In controls, lymph nodes are more numerous and smaller-sized, visible both intra- and retroperitoneally (B and D, arrowheads).

Fig. 3.

Architecture of the thymus and spleen in a SCID patient (P10) 3.5 mo after HLA-identical HSCT. (A) A normal lobular architecture and the presence of corticomedullary differentiation in the thymus, with dense cortical areas and paler medullary areas; hematoxylin and eosin (H&E), magnification 20×. (B) A normal thymus T cell pattern, with more intense CD3 staining in the medullary areas, magnification 20×. (C) Normal architecture of the spleen, with an alternation of white pulp (containing secondary B cell follicles) and red pulp; H&E, magnification 20×. (D) A normal B cell pattern in the spleen, showing B cell follicles in the white pulp and a few B cells in the red pulp; anti-CD20, magnification 20×. (E) A normal T cell pattern, showing a T cell population around the arterioles in the white pulp; anti-CD3, magnification 20×.

Fig. 4.

Architecture of gut lymphoid structures in a SCID patient (P10). (A) Transversal section of the jejunum showing the normal villous architecture of the mucosa and the absence of Peyer’s patches; H&E, magnification 20×. Note some autolysis due to autopsy. (B) Presence of very few interstitial B cells in the lamina propria of the jejunum and absence of follicles; anti-CD20, magnification 100×. (C) Presence of very few interstitial T cells in the lamina propria of the jejunum, anti-CD3, magnification100×. (D) Control jejunum showing normal villous architecture and numerous lymphoid cells within the lamina propria, including follicles (F); H&E, magnification 40×. (E) Transversal section of the appendix (P10), containing large B cell follicles in the lamina propria with well-defined germinal centers (GC) with mantle cell hyperplasia (M), H&E, magnification 40×. (F) Intense anti-CD20 staining of hyperplastic follicles; anti-CD20, magnification 40×. (G) Interfollicular T cell areas anti-CD3, magnification 40×. (H) Control appendix showing follicles with mantle (M) and prominent germinal centers (GC) in the lamina propria, magnification 40×. (I) Control appendix intense anti CD20 staining underlying follicles, anti-CD20, magnification 40×. (J) Control appendix inter follicular T cell areas and presence of a minority of intrafollicular T cells, anti-CD3, magnification 40×.

Fig. 5.

Architecture of lung TLOs in P10, and a control non-SCID case with bronchiectasis. (A) A cross-section of a suppurative dilated bronchial structure from P10 with a fungus ball in the center and many neutrophils in the lumen and in the thick inflamed mucosa, surrounded by a large number of follicles; H&E, magnification 40×. (B) A large number of B cell follicles at the edge of the suppurative, organized bronchial wall; anti-CD20, magnification 40×. (C) Interfollicular T cells in peribronchial areas from P10 with a suppurative bronchial infection; anti-CD3, magnification 40×. (D) A cross-section of postinfectious bronchiectasis in a control (non-SCID) individual with bronchial lumen dilation and inflamed bronchial and peribronchial walls; H&E, magnification 40×. (E) Small B cell lymphoid structures in the bronchial wall of a control (non-SCID) individual with bronchiectasis; anti-CD20, magnification 100×. (F) Interspersed T cells within the bronchial wall in a control (non-SCID) individual with bronchiectasis; anti-CD3, magnification 100×.

Fig. 6.

Architecture of peribronchial TLOs in P11. (A) Inflamed bronchial wall in a suppurative dilated bronchial area; H&E, magnification 20×. (B) Numerous T cells in the bronchial wall; anti-CD3, magnification 20×. (C) Numerous lymphoid follicles in the inflamed bronchial wall; anti-CD20, magnification 20×. (D) Presence of a follicular dendritic meshwork within the lymphoid follicle in the inflamed bronchial wall; anti-CD21, magnification 100×. (E) Absence of NKP46⁺ cells in the bronchial epithelium; anti-CD21, magnification 40×.

Fig. 7.

Absence of RORC⁺ CD3⁻ cells in the appendix and peribronchial lymphoid structures in P10 and P11, and presence of LTα⁺ and RANKL⁺ cells. (A) P10’s appendix: absence of RORC⁺ CD3⁻ cells in the lamina propria, and the presence of CD3⁺ T cells; double staining with anti-RORC (Brown) and anti-CD3 (red), magnification 200×. (B) The control’s appendix: the presence of RORC⁺CD3⁻ cells in the lamina propria (arrows); double staining with anti-RORC (brown), anti-CD3 (red), magnification 200×. (C) P10’s peribronchial lymphoid structure: presence of a numerous CD3⁺ T cells without RORC⁺ CD3⁻ cells; double staining with anti-RORC (brown) and anti-CD3 (red), magnification 400×. (D) P11’s peribronchial lymphoid structure: the presence of a large number of CD3⁺ T cells but no RORC⁺ CD3⁻ cells with the exception of single cell (arrow); double staining with anti-RORC (brown) and anti-CD3 (red), magnification 400×. (E) The control’s peribronchial lymphoid structure: presence of RORC⁺CD3⁻ cells in the TLOs (arrows), double staining with anti-RORC (brown), anti-CD3 (red), magnification 400×. (F) P10’s LTα⁺ TLOs in the lung: the presence of few intracytoplasmic Ltα⁺/RORC⁻ cells in the peribronchial area and the absence of RORC⁺ cells; double staining with anti-RORC (red) and anti-LTα (brown), magnification 200×. (G) The control’s LTα⁺ TLOs in the lung: the presence of intracytoplasmic Ltα⁺ cells in the peribronchial area and the presence of numerous RORC⁺ cells with red nuclear staining, double staining with anti-RORC (red), anti-LTα (brown), magnification 200×. (H) P10’s RANKL⁺ TLOs in the lung: the absence of RORC⁺ cells and presence of RORC-RANKL⁺ intracytoplasmic epithelial lining cells; double staining with anti-RORC (brown) and anti-RANKL (red), magnification 200×. (I) The control’s RANKL⁺ TLOs in the lung: the presence of numerous RORC⁺ RANKL⁻ lymphocytes and RORC⁻ RANKL⁺ epithelial lining cells; double staining with anti-RORC (brown) and anti-RANKL (red), magnification 200×. (J) P10’s RANKL⁺ structures in the appendix: the presence of RANKL⁺ intracytoplasmic cells in the upper part of the lamina propria; anti-RANKL, magnification 200×. (K) The control’s RANKL⁺ structures in the appendix: the presence of RANKL⁺ RORC⁻ intracytoplasmic cells in the upper part of the lamina propria, and RORC⁺ RANKL⁻ lymphoid cells; double staining with anti-RORC (brown) and anti-RANKL (red).