Sarcoidosis is a systemic granulomatous disease mainly affecting the lungs and hilomediastinal lymph nodes. It is characterized by non-caseating epithelioid cell granulomas in lymph nodes and lungs. Our study aimed to evaluate and compare T, B and NK cell subsets in the alveolar compartment, lymph nodes and the bloodstream simultaneously in the same patients to elucidate the immune responses associated with the development and progression of sarcoidosis. A secondary aim was to evaluate the distribution of CD45RA-expressing cells in the different anatomical compartments. Patients suspected to have sarcoidosis and who underwent bronchoscopy with bronchoalveolar lavage (BAL), lung-draining lymph node (LLN) biopsy by EBUS-TBNA and peripheral blood (PB) sampling were included in the study. They were monitored at the Regional Referral Centre of Siena University Hospital and the Respiratory Diseases Unit of Perugia Hospital. Multicolour flow cytometry analysis through FASCLyric was performed to assess T, B and NK cell subsets. Thirty-two patients (median age (IQR) 57 (52–58) years) were consecutively and prospectively enrolled. Machine learning analysis created a model which selected CD56dim16bright, CD8, Tfc, Th17, Th12, Tfh17, Tfh2, TcemRA, ThemRA, T naïve, Tc naïve, Breg, CD1d+CD5+, Th-reg, Tfh, Th1 and CD4 cells with an accuracy of 0.9500 (kappa 0.8750). Comparative analysis found 18 cell populations that differed significantly between the three anatomical compartments. The bloodstream was enriched in ThemRA (p= 0.0416), Tfh2 (p= 0.0189), Tfh17 (p= 0.0257), Th2 (p= 0.0212), Th17 (p= 0.0177), Th-naïve (p= 0.0368), CD56dimCD16bright (p< 0.0001), CD8 (p= 0.0319), TcemRA (p< 0.0001) and Tfc cells (p= 0.0004) compared with the alveolar compartment, while Th-reg were lower in PB than BAL (p= 0.0329). The alveolar compartment was enriched in Breg (p= 0.0249) and CD1d+CD5+(p= 0.0013) with respect to LLN samples and PB. Conversely, Tfh (p= 0.0470), Th1 (p= 0.0322), CD4 (p= 0.0486) and Tc-naïve (p= 0.0009) were more abundant in LLN than in BAL and PB. It has been speculated that changes in the relative contents of PB cells could be related to changes in production and to the selective redistribution of PB cells to granulomatous foci. This study further supports the fact that sarcoidosis is multisystemic in nature. However, the low level of immune cells in peripheral blood of patients with sarcoidosis is concerning. A re-expression of CD45RA on CD4+and CD8+cells could result in a reduction in peripheral immune activity. Thus, changes in the spectrum of the bloodstream may reflect both pathogenic and compensatory processes.