Before the homing of metastatic cancer cells, professional cancer antigen-presenting cells, i.e., interdigitating dendritic cells (DCs) and macrophages, seem to increase in number and change their intranodal localization (Aoki et al., 2023, Aoki et al., 2025a, Sonoda et al., 2025). Our group has noted a DC phenotype expressing DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin or CD209) as well as CD169 (sialoadhesin)-positive macrophages because of the cross-presentation of cancer antigens between these populations to induce T-lymphocyte activation (Grabowska et al., 2018). These major players for cancer antigen presentation seemed to be activated or suppressed in the pulmonary nodule or cancer lesion and they likely migrate through lymph vessels to accumulate in the subcapsular sinus of the regional nodes. CD169-positive macrophages are also known as a major player of anti-virous immunity (Bouma et al., 2025). However, there seems to be no basic information such as 1) DCs in the nodule and 2) lymph vessels associated with the nodule. Therefore, we reexamined peripheral lymphatics of the lung at a site distant from the early cancer lesion as well as at another site near the cancer. Because both types of specimens were largely composed of alveoli (or alveolar lobules) and bronchioles, below, we use the term “alveolar region” instead of the “intralobular, interlobular and subpleural region with a small volume of peripheral bronchi”.

Fig. 1 shows possible courses of lymph vessels in the alveolar region. Most lymph vessels most likely take an intralobular course along the bronchoarterial tree or the terminal arteriole in the peripheral side of the terminal bronchiole (Kambouchner and Bernaudin, 2009, Sozio et al., 2012, Weber et al., 2018, Egashira et al., 2025), while interlobular lymph vessels also exist and they drain into the subpleural lymph vessels (Weber et al., 2018, Egashira et al., 2025). Notably, Kambouchner and Bernaudin (2009) and Egashira et al. (2025) demonstrated intralobular lymph vessels running though the interalveolar septum “without” any concomitant arteriole. The first aim of this study was to “connect” those lymph vessels to a lymph nodule in the alveolar region. For the aim, we needed to re-evaluate the intra- and inter-lobular lymphatic anatomy because of no nodules in the classical scheme.

Richmond et al. (1993) strictly discriminated the bronchus-associated lymphatic tissue (BALT) from a “non-specific aggregation” of lymphocytes along arterioles. However, in the present study, a pulmonary nodule was simply defined as a mononuclear cell cluster over 50 µm in thickness. We hypothesized three types of pulmonary lymph nodules: a bronchiole-associated nodule, an arteriole-associated nodule and a subpleural nodule (Fig. 1). In this context, some or most of the bronchiole-associated nodule may correspond to BALT although the latter concept is based on antigen uptake through the bronchial epithelium, not from the afferent lymph vessel (Pabst and Tschernig, 2010). Kawamata et al. (2009) found BALT in 17 of 45 lung specimens obtained by lobectomy for lung cancer (35%). Thus, the present specimens likely contained BALT. However, because of the relatively small frequency (=35%), the arteriole-associated nodule and subpleural nodule may be predominant, rather than the bronchiole-associated nodule and BALT. The second aim was to describe composite cells of the nodule, especially DCs and CD169-positive macrophages those are major players of cancer antigen presentation (introduced in the first paragraph).

Pulmonary lymph nodules had been reported in normal lungs without tumor or inflammation: e.g., lung for donation of transplants in Richmond et al. (1993); children’s lung in Hiller et al. (1997). However, in the human lung alveolar region, DC-SIGN-positive DCs and CD169-positive macrophages have been reported only in pathological conditions: DC-SIGN-, CD83-, and/or CD1a-positive DCs in chronic obstructive pulmonary disease (van Pottelberge et al., 2010, Zanini et al., 2014, Mori et al., 2023) and pulmonary hypertension (Perros et al., 2007); and CD169 expression under respiratory syncytial virus infection (Oh et al., 2017). Moreover, there is no information in which site DCs and CD169-positive cells exist: in the nodule, subepithelial tissue or others. Adult alveolar macrophages are considered to perform de novo self-renewal (reviewed by Misharin et al. 2017). Anthracotic alveolar macrophages (macrophages containing carbon particles) are CD68-positive but CD169-negative (Aoki et al., 2025a). Therefore, CD169-positive cells represent non-alveolar, bone marrow-derived macrophages in the present study