# CD4/CD8 T-cell ratio in bronchoalveolar lavage fluid as a marker of sarcoidosis severity: a retrospective study.

Thibaut Zannese, Jean-François Viallard, Soumaya Sridi-Cheniti, Camille Prot-Leurent, Henry Dupuy, Cédric Léonard, Pierre Duffau, Estibaliz Lazaro, Carine Greib, Etienne Rivière

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Thibaut Zannese, Jean-François Viallard, Soumaya Sridi-Cheniti, Camille Prot-Leurent, Henry Dupuy, et al.. CD4/CD8 T-cell ratio in bronchoalveolar lavage fluid as a marker of sarcoidosis severity: a retrospective study.. BMC Pulmonary Medicine, 2025, 25 (1), pp.227. 10.1186/s12890-024-03428-5. hal-05086058

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# CD4/CD8 T-cell ratio in bronchoalveolar lavage fluid as a marker of sarcoidosis severity: a retrospective study

Thibaut Zannese<fnref n="1" />, Jean-François Viallard<fnref n="1" />,<fnref n="2" />†, Soumaya Sridi-Cheniti<fnref n="4" />†, Camille Prot-Leurent<fnref n="1" />, Henry Dupuy<fnref n="1" />, Cédric Léonard<fnref n="1" />, Pierre Duffau<fnref n="3" />,<fnref n="5" />, Estibaliz Lazaro<fnref n="1" />,<fnref n="3" />, Carine Greib<fnref n="1" />*† and Etienne Rivière<fnref n="1" />,<fnref n="2" />*†

**Abstract**

**Background** Sarcoidosis is a complex multisystemic disease with a wide range of clinical manifestations. Its severity and evolution are highly variable and the prognostic utility of paraclinical markers is debated. We analysed the utility of the CD4/CD8 T-cell ratio (R) in bronchoalveolar lavage (BAL) fluid as a marker of sarcoidosis severity.

**Methods** We conducted an observational, retrospective, monocentric study involving patients with systemic sarcoidosis who underwent BAL for diagnostic purposes between June 2010 and April 2020.

**Results** Among 62 patients, half had a CD4/CD8 T-cell ratio ($R$) value ≥ 3.5. Patients with $R < 3.5$ had more extrapulmonary manifestations, mainly abdominal (32.3% vs. 6.5%, $p = 0.01$) and tended to have a higher frequency of cardiac/ central nervous system involvement (19.4% vs. 6.5%, $p = 0.25$). They also had more frequent interstitial lung involvement (80.6% vs. 67.7%, $p = 0.2$) and tended to have more severe respiratory impairment. The activation ($p = 0.01$) of CD8+T cells in peripheral blood was significantly higher in patients with $R < 3.5$. By contrast, patients with $R \ge 3.5$ tended to have more frequent musculoskeletal–cutaneous involvement (48.4% vs. 32.3%, $p = 0.2$). Treatment was initiated more frequently in patients with $R < 3.5$ (71% vs. 35.5%, $p = 0.01$), who also more frequently required immunosuppressive agents (54.5% vs. 36.4%, $p = 0.02$).

**Conclusion** An $R$ value < 3.5 in BAL fluid has potential as a marker of sarcoidosis severity. Patients with a low $R$ value had a worse prognosis with more severe respiratory impairment and more frequent multisystemic and extra-pulmonary involvement, and more frequently required immunosuppressive agents.

**Keywords** Sarcoidosis, Bronchoalveolar lavage, CD4/CD8 ratio, Lymphocytic alveolitis

<footnote>† Jean-François Viallard, Soumaya Sridi-Cheniti, Etienne Rivière and Carine Greib these authors contributed equally to this work.</footnote>

<footnote>*Correspondence:</footnote>

<footnote>Carine.Greib@chu-bordeaux.fr
Etienne.Rivière
Etienne.riviere@u-bordeaux.fr</footnote>

<footnote n="1">¹ Internal Medicine and Infectious Diseases Unit, Haut-Leveque Hospital, University Hospital Centre of Bordeaux, Pessac Cedex 33604, France</footnote>

<footnote n="2">² INSERM U1034, Bordeaux University, Pessac Cedex 33604, France</footnote>

<footnote n="3">³ UMR CNRS 5164, ImmuonoconcEpT & FHU ACRONIM, Bordeaux University, Bordeaux 33000, France</footnote>

<footnote n="4">⁴ Department of Cardio-Vascular Imaging, CHU Bordeaux, University Bordeaux, Bordeaux 33000, France</footnote>

<footnote n="5">⁵ Internal Medicine Department, Saint-André Hospital, University Hospital Centre of Bordeaux, Bordeaux 33600, France</footnote>
## Introduction

Sarcoidosis is a multisystemic inflammatory disease characterised by the formation of immune non-caseating granulomas in several organs. Sarcoidosis can be severe, leading to death in approximately 7% of cases with lung and heart involvement [1, 2], or to severe disability when the central nervous system is damaged (~5% of patients) [3, 4]. Although progress has been made in understanding the mechanisms of the disease, its physiopathology is incompletely understood. A dysregulated immune response against unknown environmental antigens in a genetically susceptible background leads to granulomatous inflammation and failure to clear the causative antigen [5–7]. The disease predominantly affects the lungs and intrathoracic lymph nodes (80–90%) but can involve any organ (with or without lung involvement). Consequently, its clinical presentation is highly variable. The prognosis of sarcoidosis ranges from spontaneous remission to chronic inflammation, fibrosis, or irreversible organ failure. Except in Löfgren and Heerfordt syndromes, the diagnosis can be challenging because of the clinical diversity and its mimicry of other disorders such as tuberculosis, lymphoma, or histiocytoses. Furthermore, despite similar clinical and radiological presentations, and clear differential diagnoses, a non-caseating granuloma can be missed in biopsy samples.

Because the lungs and intrathoracic lymph nodes are frequently affected, flexible bronchoscopy with bronchial biopsies and bronchoalveolar lavage (BAL) aspiration is useful in many cases of sarcoidosis [8, 9]. In sarcoidosis, analysis of BAL fluid shows a moderately elevated lymphocyte proportion (20–50%) in 80% of cases, but this is nonspecific because it can be seen in other granulomatous or interstitial lung diseases [10, 11]. The CD4/CD8 T-cell ratio (R) in BAL fluid is increased as a result of excessive CD4⁺ T-cell proliferation [12, 13]; a cut-off R value of 3.5 has been suggested to be diagnostic of sarcoidosis [14, 15]. However, this ratio has highly variable sensitivities and specificities: some patients with sarcoidosis have R < 3.5, and R > 3.5 has been linked to other diseases such as idiopathic pulmonary fibrosis and granulomatous diseases (hypersensitivity pneumonitis, lymphoma, and tuberculosis) [16–19].

Paraclinical prognostic markers of the chronicity and severity of sarcoidosis are not available, a purpose for which the R value in BAL fluid has potential. The R value reportedly has utility as a marker of severity, and a low R value has been associated with severe lung disease [19, 20]. We evaluated the utility of the R value as a marker of the severity of sarcoidosis.

## Patients and methods

### Study design and patients

We conducted an observational, monocentric, retrospective study in the Internal Medicine Department of Bordeaux University Hospital. The study population comprised patients who had undergone flexible bronchoscopy with BAL in the preceding decade. The inclusion criterion was a diagnosis of sarcoidosis by immunological analysis of BAL fluid. The exclusion criteria were diagnosis of sarcoidosis before BAL, no immunological analysis of BAL fluid, or a diagnosis other than sarcoidosis.

### Data collection

Anonymised data on sex, age at BAL, clinical signs, pulmonary function tests (PFTs), and diffusing capacity of the lung for carbon monoxide (DLCO) were retrospectively extracted from the electronic medical records. We also evaluated the plasma level of angiotensin-converting enzyme (ACE) and the phenotypes of B and T cells in peripheral blood. Therapeutic interventions after BAL and the diagnosis of sarcoidosis were documented, including steroids, methotrexate, TNF inhibitors, mycophenolate mofetil, and cyclophosphamide.

### Diagnostic criteria and paraclinical analyses

Sarcoidosis was diagnosed according to the WASOG criteria as a clinical phenotype suggestive of sarcoidosis, confirmed by a biopsy showing non-caseating epithelioid granulomas, with any differential diagnosis excluded [21, 22]. In some cases, sarcoidosis was evaluated as highly probable if granulomas were not detected in biopsy samples.

The patients were divided into five groups based on their clinical phenotypes according to the Genotype–Phenotype Relationship in Sarcoidosis (GenPhenReSa) study [23]: group 1 with pulmonary and intrathoracic lymph node involvement, group 2 with musculoskeletal–cutaneous involvement, group 3 with abdominal organ involvement (kidneys, spleen, liver, and digestive tract), group 4 with ocular–cardiac–cutaneous–central nervous system (CNS) involvement, and group 5 with extrapulmonary involvement.

Pulmonary function was considered to be altered in the presence of restrictive (forced vital capacity < 80%) or obstructive (forced expiratory volume in one second÷forced vital capacity < 0.7) pulmonary disease.

Computed tomography (CT) scans were analysed by chest radiologists in terms of a correlation between the R value in BAL fluid and the extension of micronodular lesions (sparse or profuse, galaxy sign) and/or fibrotic damage. The CT scan analysis was descriptive, aiming to find a correlation between the extension of interstitial
involvement related to sarcoidosis and the CD4/CD8 ratio. The items were simplified by referring to the CT activity score proposed by Benamore R et al. in 2016 [24]. Micronodular involvement affecting less than 25% of the lung parenchyma is described as sparse, while involvement affecting more than 25% is described as profuse. Consolidations/conglomerates and traction bronchiectasis were noted separately, as they could indicate more advanced involvement.

Lymphocyte subsets were analysed in the Bordeaux University Hospital Laboratory of Immunology using a Beckman-Coulter FC500 Flow Cytometer as described previously [25]. This yielded the total lymphocyte count and the percentages and numbers of CD3⁻ (mainly B and NK cells), CD19⁺ B cells, CD3⁺ T cells, CD4⁺ T-helper cells, CD8⁺ cytotoxic T cells, CD3⁺CD4⁻CD8⁻ cells, CD3⁺HLADR⁺ activated T cells, CD3⁺CD4⁺HLADR⁺ activated T-helper cells, and CD3⁺CD8⁺HLADR⁺ activated cytotoxic T cells.

## Outcomes

The primary outcomes were differences in the clinical, paraclinical, and radiological profiles of patients diagnosed with sarcoidosis based on predefined R cut-off values ($R \ge 3.5$ or $< 3.5$). The secondary outcomes were the following:

* Correlations among ACE levels, lymphocyte phenotypes, and the R value in BAL fluid.

* Treatments administered based on predefined cut-off R values ($R \ge 3.5$ or $< 3.5$).

## Statistical analysis

Descriptive analyses are shown as counts (percentages), means and standard deviations (SD), or medians and interquartile ranges (IQR). To test for independence between two categorical variables, the chi-squared test was used (or Fisher's exact test if theoretical counts were < 5). The Mann-Whitney non-parametric test was used to compare medians, and Student's t-test to compare means. A two-sided value of $p < 0.05$ was considered indicative of statistical significance. Spearman's test was used to assess correlations between two variables. Analyses were performed using SPSS 26 software (IBM SPSS Statistics).

## Ethical considerations

Because this retrospective study used anonymised healthcare data, the necessity for patient consent was obviated in accordance with French legislation. In compliance with French statutory regulations, use of the data for retrospective research was reported to the Data

Protection Officer at the University Hospital of Bordeaux, and acknowledgment of the use of anonymised data was obtained. As mandated, patients were informed of the use of their anonymised data by means of a departmental booklet. The Institutional Review Board of our institution approved this study (MR-004, French Government).

## Results

### Patients' characteristics

Between June 2010 and April 2020, flexible bronchoscopy with BAL fluid aspiration was performed on 451 patients, among whom 207 underwent immunological analyses including measurement of the R value. Seventy-six patients had sarcoidosis, including 14 being followed for sarcoidosis before BAL, who were therefore excluded; 62 patients were included in the study (Fig. 1). Among the 62 patients, 31 (50%) had $R \ge 3.5$. The median age and the sex distribution at BAL were similar in the two groups (Table 1). Typical non-caseating granuloma was found in 50 patients (80.6%); the other 12 patients had overt clinical and radiological features of sarcoidosis and no differential diagnosis. Granuloma was found in endobronchial ($n=25$), surgical lymph node ($n=13$), or salivary gland ($n=9$) biopsies. Among the 16 patients who did not have lung damage and underwent bronchial biopsies, granulomas were found in 1 patient (6.3%), compared to 25 (54.3%) of the 46 patients with interstitial lung damage.

### Patient profiles according to CD4/CD8 ratio (R) value

#### Clinical profiles

All the 62 patients had interstitial lung involvement, enlarged intrathoracic lymph nodes, or both. Intrathoracic lymph nodes were present in all patients with $R \ge 3.5$ ($n=31$) and in 96.8% of those with $R < 3.5$ ($n=30$). Parenchymal lung involvement tended to be more frequent in patients with $R < 3.5$ ($n=25$, 80.6% vs. $n=21$, 67.7%, $p=0.2$; Table 1), and only 13 patients had isolated lung involvement: 7 in the $R \ge 3.5$ group (22.6%) and 6 in the $R < 3.5$ group (19.4%). Among the 12 patients with abdominal organ involvement, 10 (32.3%) had $R < 3.5$ and 2 (6.5%) had $R \ge 3.5$ ($p=0.02$). Among the 12 patients in the OCCC group, 6 had $R \ge 3.5$ (2 with cardiac and/or CNS involvement and 4 with isolated eye lesions). All the six patients in the $R < 3.5$ group had cardiac and/or CNS disease involvement and none had ocular involvement. In this group, skin lesions were systematically associated with cardiac and/or CNS involvement. Finally, four patients had extrapulmonary involvement (two in each group) and all had intrathoracic and extrathoracic enlarged lymph nodes with no other extrapulmonary manifestation, except for one patient in the $R \ge 3.5$ group with ear-nose-throat involvement.
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Flow chart of the study population. Abbreviations: R, CD4/CD8 ratio; BAL, bronchoalveolar lavage</figcaption></figure>

<table><caption>**Table 1** Clinical characteristics of patients with R values ≥ 3.5 and < 3.5</caption><thead><tr><th>Characteristic n (%)</th><th>Total<br>n=62</th><th>R ≥ 3.5<br>n=31</th><th>R < 3.5<br>n=31</th><th>p-Value</th></tr></thead><tbody><tr><td>Median age at BAL (range)</td><td>43.5 (19-72)</td><td>43 (19-72)</td><td>44 (23-68)</td><td>0.921</td></tr><tr><td>Sex [n (%)]</td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>35 (56.5)</td><td>17 (54.8)</td><td>18 (58.1)</td><td>0.798</td></tr><tr><td>Female</td><td>27 (43.5)</td><td>14 (45.2)</td><td>13 (42)</td><td></td></tr><tr><td>Intrathoracic lymph node [n (%)]</td><td>61 (98.4)</td><td>31 (100)</td><td>30 (96.8)</td><td>1.000<sup>5</sup></td></tr><tr><td>Parenchymal involvement [n (%)]</td><td>46 (74.2)</td><td>21 (67.7)</td><td>25 (80.6)</td><td>0.246</td></tr><tr><td>Phenotypes [n (%)]</td><td></td><td></td><td></td><td></td></tr><tr><td>Isolated lung involvement</td><td>13 (20.9)</td><td>7 (22.6)</td><td>6 (19.4)</td><td>0.755</td></tr><tr><td>Musculoskeletal-cutaneous</td><td>25 (40.3)</td><td>15 (48.4)</td><td>10 (32.3)</td><td>0.196</td></tr><tr><td>Abdominal involvement</td><td>12 (19.4)</td><td>2 (6.5)</td><td>10 (32.3)</td><td><strong>0.02</strong><sup>5</sup></td></tr><tr><td>“OCCC”</td><td>12 (19.4)</td><td>6 (19.4)</td><td>6 (19.4)</td><td>1.000<sup>5</sup></td></tr><tr><td>Ocular</td><td>4 (6.5)</td><td>4 (12.9)</td><td>0 (0)</td><td>0.113<sup>5</sup></td></tr><tr><td>CCC</td><td>8 (12.9)</td><td>2 (6.5)</td><td>6 (19.4)</td><td>0.255<sup>5</sup></td></tr><tr><td>Extrapulmonary involvement*</td><td>4 (6.5)</td><td>2 (6.5)</td><td>2 (6.5)</td><td></td></tr></tbody></table>

Abbreviations: R CD4/CD8 ratio, BAL bronchoalveolar lavage, OCCC ocular-cardiac-cutaneous-central nervous system, CCC cardiac-cutaneous-central nervous system

## Respiratory outcomes

The median cell count in BAL fluid was 160,000/mL, comprising 24.5% lymphocytes, 69% macrophages, 0.6% eosinophils, and 5.2% neutrophils. The median R value was 3.45 (range 0.48–45.71) (Table 2). There was a tendency for more impaired pulmonary function in patients with R < 3.5; 51.6% had pathological PFT results compared to 35.4% in the R ≥ 3.5 group (p=0.09). The DLCO tended to be lower in patients with R < 3.5 (median 68% vs. 81% in those with R ≥ 3.5; p=0.057) (Table 2).

Because patients with R < 3.5 had more severe disease, we evaluated the correlation of the R value with CT findings. Among the 46 patients with parenchymal involvement, we were able to review the CT scans at diagnosis of only 29 (13 with R ≥ 3.5 and 16 with R < 3.5; Table 2), because we had no access to the detailed radiological sections of the CT scans of the other 17 patients. The frequency of micronodular involvement (sparse and pro-fuse) was similar in the two groups. One patient, who had R < 3.5, had fibrosis.

<footnote>* All had intra- and extra-pulmonary node involvement without other extrapulmonary manifestations, except for one with otorhinolaryngologic involvement (in the R ≥ 3.5 group)</footnote>

<footnote n="5">⁵ Fishers' test with exact p-value</footnote>
<table><caption>**Table 2** Respiratory findings according to R value</caption><thead><tr><th>Characteristics n (%)</th><th>Total<br>n = 62</th><th>R ≥ 3.5<br>n = 31</th><th>R < 3.5<br>n = 31</th><th>p-value</th></tr></thead><tbody><tr><td><strong>BAL's data</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total cell count (× 10<sup>3</sup>/ml) [median (IQR)]</td><td>160 (100–250)</td><td>145 (97–235)</td><td>160 (100–275)</td><td>0.471</td></tr><tr><td>Cell population % mean (SD)</td><td></td><td></td><td></td><td></td></tr><tr><td>Lymphocytes</td><td>24.5 (15.9)</td><td>28.4 (15.7)</td><td>20.8 (15.5)</td><td>0.071</td></tr><tr><td>Macrophages</td><td>69 (5.2)</td><td>66.3 (14.8)</td><td>71.7 (15.3)</td><td>0.184</td></tr><tr><td>Eosinophils</td><td>0.6 (1.8)</td><td>0.7 (1.8)</td><td>0.6 (1.7)</td><td>0.732</td></tr><tr><td>Neutrophils</td><td>5.2 (7.1)</td><td>4.7 (8.1)</td><td>5.7 (6.0)</td><td>0.599</td></tr><tr><td>CD4/CD8 ratio [median (IQR)]</td><td>3.45 (1.88–7.26)</td><td>6.84 (4.78–11.32)</td><td>1.91 (1.28–2.81)</td><td><strong>&lt;0.001</strong></td></tr><tr><td><strong>PFTs data</strong></td><td><strong>n = 59</strong></td><td><strong>n = 31</strong></td><td><strong>n = 28</strong></td><td></td></tr><tr><td>FEV-1 (L) [median (IQR)]</td><td>2.94 (2.34–3.56)</td><td>3.07 (2.57–3.74)</td><td>2.75 (1.84–3.42)</td><td>0.069</td></tr><tr><td>FVC (L) [median (IQR)]</td><td>3.8 (2.92–4.48)</td><td>3.89 (3.24–5.01)</td><td>3.53 (2.47–4.15)</td><td>0.092</td></tr><tr><td>DLCO (%) [median (IQR)]</td><td>76 (66–87)</td><td>81 (70–86)</td><td>68 (60–88)</td><td>0.057</td></tr><tr><td><strong>CT-scan review [median (IQR)]**</strong></td><td><strong>n = 29</strong></td><td><strong>n = 13</strong></td><td><strong>n = 16</strong></td><td></td></tr><tr><td>Micronodular involvement [n (%)]</td><td></td><td></td><td></td><td></td></tr><tr><td>Sparce</td><td>13 (44.8)</td><td>5 (38.5)</td><td>8 (50.0)</td><td>0.534</td></tr><tr><td>Profuse</td><td>7 (24.1)</td><td>3 (23.1)</td><td>4 (25.0)</td><td>1.000<sup>§</sup></td></tr><tr><td>Consolidation/conglomeration</td><td>9 (31.0)</td><td>5 (38.5)</td><td>4 (25.0)</td><td>0.688<sup>§</sup></td></tr><tr><td>Traction bronchiectasis [n (%)]</td><td>1 (3.4)</td><td>0 (0)</td><td>1 (6.3)</td><td>1.000<sup>§</sup></td></tr></tbody></table>

Consolidation: Homogeneous increase in pulmonary parenchymal attenuation which obscures vessels and airway walls; Conglomeration: a large opacity > 3 cm surrounding and encompassing hilar bronchi and vessels; Traction bronchiectasis: irregular bronchial dilatation due to surrounding fibrosis. Definition of terms for abnormalities on HRCT from Fleischner Glossary [26]

Abbreviations: BAL Bronchoalveolar lavage, PFTs Pulmonary function tests, FEV-1 Forced expiratory volume, FVC Forced vital capacity, DLCO Diffusing capacity of the lung for carbon monoxide, IQR interquartile range, SD standard deviation

* Restrictive pulmonary disease and/or obstructive pulmonary disease

** CT-scans review for sarcoidosis patients with parenchymal involvement was available for 29 patients (13 with R ≥ 3.5 and 16 with R < 3.5)

§ Fisher Test with exact p-value

## Biological characteristics

We analysed the patients' biological characteristics at diagnosis according to CD4/CD8 ratio value (Table 3). The ACE level tended to be higher in the ratio ≥ 3.5 group (median 73 IU/L vs. 54.5 IU/L in the ratio < 3.5 group; *p* = 0.082). The plasma B- and T-cell counts were similar in the two groups. The number of CD8+ T cells tended to be greater in the ratio < 3.5 group (median 0.29 vs. 0.19 × 10⁹/L in the ratio ≥ 3.5 group; *p* = 0.06). The number of activated CD8+ T cells was significantly higher in patients with ratio < 3.5 (median 0.07 × 10⁹/L [32.8% of total CD8+ T cells] vs. 0.03 × 10⁹/L [21.1% of total CD8+ T cells] in patients with ratio ≥ 3.5 [*p* = 0.014]). Patients with a high percentage of activated CD8+ T cells in peripheral blood had a lower ratio value in BAL fluid (R = -0.3, *p* = 0.03). Importantly, a ratio < 3.5 was correlated with a high percentage of CD8+ T cells (R = -0.27, *p* = 0.04), but there was no such correlation with the percentage of CD4+ T cells (R = -0.17, *p* = 0.22). Patients with a high percentage of activated CD4+ T cells had a high ACE level (R = 0.35, *p* = 0.01), but this was not the case for those with a high percentage of activated CD8+ T cells (R = 0.05, *p* = 0.7).

## Therapeutic management

Among the 62 patients in whom sarcoidosis was diagnosed, 33 underwent treatment: 11 in the R ≥ 3.5 group (35.5%) and 22 in the R < 3.5 group (71%) (*p* = 0.01; Table 4). Initiation of treatment was indicated in cases of parenchymal damage with pathological PFT results or extrapulmonary involvement such as cardiac, neurological, and abdominal disorders. Steroids were used in all but one patient who had R < 3.5. An immunosuppressive agent or a biologic therapy was required for 4 patients in the R ≥ 3.5 group (36.4%) and 12 patients in the R < 3.5 group (54.5%, *p* = 0.04); these were methotrexate (n = 7), TNF inhibitor (n = 8), mycophenolate mofetil (n = 6), and cyclophosphamide (n = 3). An immunosuppressive agent was used as the first-line therapy in association with steroids in three patients in the R ≥ 3.5 group (75%) and seven in the R < 3.5 group (58.3%). Among the 16 patients who received an immunosuppressive agent or a biologic therapy, 9 received one (3 in the R ≥ 3.5 group [75%] and 6 in the R < 3.5 group [50%]), 6 received two successively (1 in the R ≥ 3.5 group [25%] and 5 in the R < 3.5 group [41.6%]), and 1 patient in the R < 3.5 group received at least three agents successively (6.3%).
<table><caption>**Table 3** Biological characteristics of patients with sarcoidosis with $R \ge 3.5$ and $R < 3.5$</caption><thead><tr><th>Characteristic n (%)</th><th>Total n = 62</th><th>R &ge; 3.5 31 (50)</th><th>R &lt; 3.5 31 (50)</th><th>Normal range</th><th>p</th></tr></thead><tbody><tr><td><strong>Biological data [median (IQR)]*</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total lymphocytes count &times; 10<sup>9</sup>/L</td><td>1.22 (0.87-1.74)</td><td>1.17 (0.80-1.60)</td><td>1.28 (0.97-1.79)</td><td>1.30-2.70</td><td>0.125</td></tr><tr><td>&mdash;B cells &times; 10<sup>9</sup>/L</td><td>0.35 (0.23-0.51)</td><td>0.32 (0.24-0.50)</td><td>0.36 (0.22-0.51)</td><td>0.29-0.80</td><td>0.987</td></tr><tr><td>&mdash;T cells &times; 10<sup>9</sup>/L</td><td>0.81 (0.54-1.25)</td><td>0.73 (0.42-1.14)</td><td>0.90 (0.60-1.27)</td><td>0.70-2.10</td><td>0.243</td></tr><tr><td>&mdash;CD4 cells &times; 10<sup>9</sup>/L</td><td>0.52 (0.31-0.83)</td><td>0.51 (0.25-0.83)</td><td>0.54 (0.37-0.86)</td><td>0.50-1.30</td><td>0.470</td></tr><tr><td>&mdash;CD8 cells &times; 10<sup>9</sup>/L</td><td>0.23 (0.14-0.37)</td><td>0.19 (0.10-0.32)</td><td>0.29 (0.19-0.43)</td><td>0.20-0.70</td><td>0.060</td></tr><tr><td>&mdash;CD4/CD8 ratio</td><td>2.10 (1.60-2.90)</td><td>2.40 (1.80-2.90)</td><td>2.00 (1.50-2.50)</td><td></td><td>0.175</td></tr><tr><td>T cells activated &times; 10<sup>9</sup>/L</td><td>0.13 (0.07-0.17)</td><td>0.09 (0.06-0.15)</td><td>0.15 (0.1-0.21)</td><td>0.03-0.27</td><td>0.152</td></tr><tr><td>&mdash;% among total lymphocytes</td><td>9.58 (6.65-16.00)</td><td>9.18 (6.74-12.36)</td><td>14.3 (6.62-20.1)</td><td>2.10-13.7</td><td>0.332</td></tr><tr><td>&mdash;% among T cell</td><td>16.20 (10.40-23.50)</td><td>13.55 (11.00-17.20)</td><td>20.50 (9.55-29.00)</td><td>2.80-17.7</td><td>0.346</td></tr><tr><td>CD4+T-cell activated &times; 10<sup>9</sup>/L</td><td>0.06 (0.04-0.10)</td><td>0.05 (0.03-0.11)</td><td>0.06 (0.05-0.10)</td><td>0.02-0.08</td><td>0.727</td></tr><tr><td>&mdash;% among total lymphocytes</td><td>5.03 (3.30-8.20)</td><td>5.01 (3.20-8.30)</td><td>5.03 (3.50-8.00)</td><td>1.00-4.30</td><td>0.836</td></tr><tr><td>&mdash;% among CD4</td><td>12.80 (8.30-17.90)</td><td>14.55 (7.00-17.70)</td><td>12.30 (10.20-18.10)</td><td>2.10-10.40</td><td>0.970</td></tr><tr><td>CD8+T-cell activated &times; 10<sup>9</sup>/L</td><td>0.05 (0.030-0.10)</td><td>0.03 (0.02-0.07)</td><td>0.07 (0.05-0.12)</td><td>0.01-0.17</td><td><strong>0.014</strong></td></tr><tr><td>&mdash;% among total lymphocytes</td><td>4.22 (2.40-8.80)</td><td>2.99 (2.20-5.70)</td><td>7.06 (3.70-11.60)</td><td>0.70-9.90</td><td>0.069</td></tr><tr><td>&mdash;% among CD8</td><td>26.28 (14.60-37.30)</td><td>21.10 (12.50-30.10)</td><td>32.8 (18.10-42.10)</td><td>3.00-33.50</td><td>0.125</td></tr><tr><td>ACE IU/L</td><td>66.0 (44.0-102.0)</td><td>73.0 (50.0-108.0)</td><td>54.5 (41.5-88.0)</td><td>20.0-70.0</td><td>0.082</td></tr></tbody></table>

Abbreviations: R CD4/CD8 T-cell ratio, IQR interquartile range, IU/L international units per litre

<table><caption>**Table 4** Therapeutic management of patients with sarcoidosis according to R value</caption><thead><tr><th>Treatment received</th><th>Total n = 62</th><th>R &ge; 3.5 n = 31</th><th>R &lt; 3.5 n = 31</th><th>p-value</th></tr></thead><tbody><tr><td>Treatment required</td><td>33 (53.2)</td><td>11 (35.5)</td><td>22 (71)</td><td><strong>0.01</strong></td></tr><tr><td>Steroids</td><td>32 (96.9)</td><td>11 (100)</td><td>21 (95.5)</td><td>1.000</td></tr><tr><td>Immunosuppressor agent</td><td>16 (48.5)</td><td>4 (36.4)</td><td>12 (54.5)</td><td><strong>0.040<sup>§</sup></strong></td></tr><tr><td>Methotrexate</td><td>7 (43.8)</td><td>2 (50)</td><td>5 (41.7)</td><td>0.425<sup>§</sup></td></tr><tr><td>TNF inhibitor</td><td>8 (50)</td><td>1 (25)</td><td>7 (58.3)</td><td>0.053<sup>§</sup></td></tr><tr><td>Mycophenolate mofetil</td><td>6 (37.5)</td><td>2 (50)</td><td>4 (33.3)</td><td>0.671<sup>§</sup></td></tr><tr><td>Cyclophosphamide</td><td>3 (18.8)</td><td>0 (0)</td><td>3 (25)</td><td>0.238<sup>§</sup></td></tr><tr><td>Lines of immunosuppressive therapy [n (%)]</td><td></td><td></td><td></td><td></td></tr><tr><td>&mdash;1</td><td>9 (56.3)</td><td>3 (75)</td><td>6 (50)</td><td>0.703<sup>§</sup></td></tr><tr><td>&mdash;2</td><td>6 (37.5)</td><td>1 (25)</td><td>5 (41.7)</td><td></td></tr><tr><td>&mdash;3</td><td>1 (6.3)</td><td>0 (0)</td><td>1 (8.3)</td><td></td></tr></tbody></table>

Abbreviation: TNF tumour necrosis factor

## Discussion

We evaluated 62 patients with sarcoidosis at diagnosis who underwent BAL with phenotyping of lymphocytes in BAL fluid. Half of the patients had $R \ge 3.5$. Patients with $R < 3.5$ had more intra-abdominal involvement and tended to have a more severe disease requiring systemic treatments and immunosuppressive agents.

Consistent with previous studies, patients with $R < 3.5$ were more likely to have parenchymal involvement than those with $R \ge 3.5$ (80.6% vs. 67.7%). Danila et al. reported a decrease in the CD4/CD8 ratio as thoracic damage increased from stage I to stage III [19, 20].

We analysed the patients' phenotypes according to the GenPheReSa study [23]. Abdominal involvement was significantly more frequent in patients with $R < 3.5$. By contrast, more patients with $R \ge 3.5$ had musculoskeletal-cutaneous involvement, likely because of the greater number of patients with Lofgren's syndrome in the $R \ge 3.5$ group. Indeed, a high CD4/CD8 ratio is reportedly a feature of Lofgren's syndrome [17, 27]. Ocular-cardiac-cutaneous-CNS involvement was equally distributed between the two groups, but the frequency of cardiac-cutaneous-CNS involvement (i.e., excluding ocular involvement) was higher in the $R < 3.5$ group. Indeed, ocular lesions were present only in patients with $R \ge 3.5$ and were isolated. Such patients are reported to be more likely to have a high R value in BAL fluid [28, 29].

$CD4^+$ T cells are typically activated in sarcoidosis and accumulate in affected organs [30, 31]. We did not find a difference between $CD4^+$ T cells activation in peripheral blood in the 2 groups, but patients with $R < 3.5$ had a larger number of activated $CD8^+$ T cells. This is consistent with prior reports that $CD8^+$ T-cell activation in peripheral blood is associated with a greater risk of more severe respiratory impairment chronic disease

<footnote>* B-cell, T-cell, and T-cell subpopulation data were available for 56 patients (27 with $R \ge 3.5$ and 29 with $R < 3.5$). T-cell activation data were available for 51 patients (26 with $R \ge 3.5$ and 25 with $R < 3.5$). ACE data were available for 55 patients (27 with $R \ge 3.5$ and 28 with $R < 3.5$)</footnote>

<footnote>§ Fisher's test with exact p-value</footnote>
progression, probably because of production of gran-
zymes, perforins, tumour necrosis factor (TNF)-α, and
interferon (INF)-γ, leading to higher cytotoxicity [32, 33].
Regarding secondary outcomes, a high ACE level tended
to be associated with a greater number of activated CD4+
T cells, and patients with R<3.5 had a high number of
circulating of CD8+ T cells, consistent with the patho-
physiology of sarcoidosis. Heightened activation of CD4+
T-helper cells is associated with the formation of more
granulomas, as reflected by the blood ACE level [13, 31].
However, during the chronic and more fibrotic phase
of the disease, the ACE level decreases with increasing
CD8+ T-cell cytotoxicity [34].

Interestingly, the treatments administered varied
according to disease severity. Patients with R<3.5 had
more extrapulmonary manifestations (excluding cuta-
neous and joint involvement) with more multisystemic
damage and required more therapeutic interventions
with an increased need for immunosuppressive agents
and multiple lines of treatment.

This study has several limitations. It was a retrospective
study involving a small number of patients, limiting the
statistical power. Additionally, the R value may be influ-
enced by factors such as disease activity, age [35], and
smoking [17, 36, 37], which we did not evaluate.

Most of our findings are consistent with prior reports;
patients with a high R value have a better prognosis [27],
and chronic disease evolution is significantly associated
with a low R value [38, 39]: high ratio correlates with
disease activity and low ratio with disease severity. Our
work retrospectively confirms these findings, as patients
with R < 3.5, especially those with abdominal or cardiac-
cutaneous–CNS involvement, had more severe disease,
required more intensive treatment and/or were refrac-
tory to successive treatment modalities, and had a high
proportion of activated CD8+ T cells in peripheral blood.

Conclusion

The R value in BAL fluid, in addition to its diagnostic
value, has potential as a marker of sarcoidosis severity.
An R value of <3.5 was strongly linked to disease sever-
ity and the requirement for more intensive treatment.
Our data provide insight into the pathophysiology of sar-
coidosis in that a low R value in BAL was associated with
CD8+ T-cell activation in peripheral blood and a poor
prognosis.

Acknowledgements

We are grateful to the patients who participated in the study and to Textcheck for editorial assistance.

Authors' contributions

TZ, JFV, SSC, EL, CG & ER were responsible for conceptualisation, methodology, writing – original draft, writing – review and editing, supervision, and project

administration. CPL, HD, CL & PD were responsible for conceptualisation, methodology, writing – review and editing.

Funding

No specific funding was received from any bodies in the public, commercial, or not-for-profit sector to carry out the work described in this article.

Data availability

The data are available upon request to the corresponding author.

Declarations

Ethics approval and consent to participate

Because this retrospective study used anonymised healthcare data, the necessity for patient consent was obviated in accordance with French legislation. In compliance with French statutory regulations, use of the data for retrospective research was reported to the Data Protection Officer at the University Hospital of Bordeaux, and acknowledgment of the use of anonymised data was obtained. As mandated, patients were informed of the use of their anonymised data by means of a departmental booklet. The Institutional Review Board of our institution approved this study (MR-004, French Government).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Received: 16 February 2024 Accepted: 29 November 2024

Published online: 09 May 2025

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