**6.2. A new insight into the pathogenesis of interstitial pneumonia in connective tissue disease: "T cells trigger interstitial pneumonia in polymyositis/dermatomyositis"**

We have speculated that exploring the early immune phases of IP in the lung would be the most direct approach to understand the pathogensis before more complex secondary immune responses occur in the evolution of IP [6, 7]. PM/DM is one of the major CTD, of which the most critical problem is pulmonary involvement. As mentioned before, ILD, mainly IP, often severe and progressive, has been recognized in 30–70% of PM/DM patients and is frequently associated with a dismal prognosis. While the presence of myositis-specific autoantibodies, such as Jo-1 and activated T cell muscle infiltrates, suggests autoimmune mechanisms in the etiology of PM, the pathogenesis of the associated IP remains undefined. We encountered two cases of early-stage PM-associated IP, of which we had an opportunity of investigating the fresh lung tissues obtained by video-assisted thoracoscopic (VATS) biopsy performed for the sake of histopathological diagnosis toward treatment options. Since this is the clear and robust demonstration of the pivotal role of T cells in CTD-IP, we herein present the details of our study with two cases of early-stage PM-associated IP both suggesting that T cells contribute to the early phase of the development of IP. Lung tissue was utilized with the approval of the institutional review board.

#### *6.2.1. Analysis in cases of interstitial pneumonia associated with polymyositis/dermatomyositis*

Patient A was a 51-year-old woman with no tobacco history and no family history of lung diseases, referred to our clinic for evaluation of arthralgia and myalgia. At 49 years, the patient noticed Raynaud's phenomenon and polyarthralgia, for which she was given low-dose prednisolone with clinical improvement. Two months before the referral, she developed polyarthralgia and myalgia. On physical examination, the patient had no skin lesions but presented with fine crackles audible on inspiration in both lower lung fields. Erythrocyte sedimentation

T cells Th1 cytokines (IFN-*Y* and IL-12) attenuate PF, Th2 cytokines (IL-4, IL-5 and IL-13) enhance PF,

Macrophages M1 marcrophages induce myofibroblast apoptosis and digest ECM by activation of MMPs. M2

Neutrophils Neutrophils produce elastase, MMPs, and TIMPs. Neutrophil elastase activates TGF-β and recruits

Fibrocytes Fibrocytes produce ECM, cross-linking enzymes, chemokines, growth factors, and MMPs, and

IL-13 IL-13 differentiates human lung fibroblast to myofibroblast through a JNK-dependent pathway.

TGF-β1 TGF-β promote EMT through SMAD-2/3 signaling pathways. TGF-β1 induces PF through ERK,

CCL2 CCL2 increase fibrocyte recruitment and differentiation into fibroblasts, resulting in excessive collagen deposition. CCL2 activates M2 macrophage activation and promote PF. CCL17 CCL17 promotes PF through the recruitment of CCR4^+ Th2 cells and alveolar macrophages. CCL18 CCL18 increase collagen production in lung fibroblasts through ERK1/2, PKCα, and Sp1/Smad3

CXCL 12 CXCL 12 recruits fibrocytes and activates the Rac1/ERK and JNK signaling pathways to induce

Definition of abbreviations: Ap-1, activator protein 1; CCL, CC chemokine; CTGF, connective tissue growth factor; CXCL, CXC chemokine ligand; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-regulated kinase; IL-1R1, IL-1 receptor 1; JNK, c-Jun N-terminal kinases; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MyD88, myeloid differentiation primary response gene 88; PDGF, plateletderived growth factor; PF, pulmonary fibrosis; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; Rac 1, Rasrelated C3 botulinum toxin substrate 1; SMAD, SMA/MAD homology; Sp1, specificity protein 1; TGF, transforming

MAPK, PI3K/Akt, and Rho-like GTPase pathways. TGF-β1 differentiates fibroblasts into and

PF. Fibroblasts can differentiate into fibroblasts and myofibroblasts.

IL-1β Profibrotic effects of IL-1β, mediated through IL-1R/MyD88 signaling pathway.

PDGF PDGF stimulates fibroblasts and increase ECM gene expression in fibroblasts.

AP-1 activation and CTGF expression in fibroblasts

growth factor; TIMP, tissue inhibitors of metalloproteinase; Tregs regulatory T cells.

and *Yδ*-T cells have an antifibrotic role in PF.

inflammatory cells to the lung, thereby promoting PF.

IL-17 IL-17 interacts/cooperates with TGF-β signaling to promote PF.

increase ECM accumulation.

signaling pathways.

**Table 5.** Possible immune mechanisms for pulmonary fibrosis.

Adapted from Kolahian et al. [158].

can exert pro- and antifibrotic effects.

Th17 cells enhance PF, Tregs and Th9 (IL-9) have both pro- and antifibrotic roles in PF; Th22 (IL-22)

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macrophages recruit and activate fibroblast through TGF-β1 and PDGF secretion. M2 macrophages further produce TIMPs and inhibit degradation of ECM. Both Macrophages phenotypes (M1/M2)

promote PF. Fibrocytes secrete paracrine mediators, which activate resident fibroblasts to promote

**Cells and mediators**

Cytokines

Chemokines

Immune cells

**Description**


Definition of abbreviations: Ap-1, activator protein 1; CCL, CC chemokine; CTGF, connective tissue growth factor; CXCL, CXC chemokine ligand; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-regulated kinase; IL-1R1, IL-1 receptor 1; JNK, c-Jun N-terminal kinases; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MyD88, myeloid differentiation primary response gene 88; PDGF, plateletderived growth factor; PF, pulmonary fibrosis; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; Rac 1, Rasrelated C3 botulinum toxin substrate 1; SMAD, SMA/MAD homology; Sp1, specificity protein 1; TGF, transforming growth factor; TIMP, tissue inhibitors of metalloproteinase; Tregs regulatory T cells.

Adapted from Kolahian et al. [158].

SSc-associated IP is rather insufficient. More fundamental investigations in this aspect are needed to address to many queries as to the whole scenario of the development of IP in CTDs. Besides, as concerns idiopathic pulmonary fibrosis (IPF), the latest evidence of immune mechanisms in IPF was reviewed in the recent literature, which includes involvement of both innate immunity and adaptive immunity at several levels of the processes toward development of fibrinogenesis in the human lung of IPF or in its model mice, as summarized in **Table 5** [158]. Briefly, in adaptive immune system, the role of T cells seems complex and subset dependent; Th2 and Th17 cells were shown to promote pulmonary fibrosis, although Th1, Th22, and γδ-T cells have been found to attenuate fibrotic disease. Treg and Th9 subsets have been proposed to exert both anti- and profibrotic effects. In innate immunity, M2 macrophages and neutrophils have been suggested to enhance pulmonary fibrosis, whereas M1 macrophages were assigned a protective role, but contradictory findings have also been

After all, a variety of studies on the pathogenesis of IPF have been conducted, and many experimental models were generated to explore the mechanisms. However, it is yet highly questionable whether the evidence provided from the studies of IPF is applicable to the etiology of CTD-associated IP. Furthermore, it is still unclear whether the animal models such as the mouse bleomycin model can truly replicate the autoimmune progressive forms of the

**6.2. A new insight into the pathogenesis of interstitial pneumonia in connective tissue disease: "T cells trigger interstitial pneumonia in polymyositis/dermatomyositis"**

*6.2.1. Analysis in cases of interstitial pneumonia associated with polymyositis/dermatomyositis*

Patient A was a 51-year-old woman with no tobacco history and no family history of lung diseases, referred to our clinic for evaluation of arthralgia and myalgia. At 49 years, the patient

We have speculated that exploring the early immune phases of IP in the lung would be the most direct approach to understand the pathogensis before more complex secondary immune responses occur in the evolution of IP [6, 7]. PM/DM is one of the major CTD, of which the most critical problem is pulmonary involvement. As mentioned before, ILD, mainly IP, often severe and progressive, has been recognized in 30–70% of PM/DM patients and is frequently associated with a dismal prognosis. While the presence of myositis-specific autoantibodies, such as Jo-1 and activated T cell muscle infiltrates, suggests autoimmune mechanisms in the etiology of PM, the pathogenesis of the associated IP remains undefined. We encountered two cases of early-stage PM-associated IP, of which we had an opportunity of investigating the fresh lung tissues obtained by video-assisted thoracoscopic (VATS) biopsy performed for the sake of histopathological diagnosis toward treatment options. Since this is the clear and robust demonstration of the pivotal role of T cells in CTD-IP, we herein present the details of our study with two cases of early-stage PM-associated IP both suggesting that T cells contribute to the early phase of the development of IP. Lung tissue was utilized with the approval of

described [158].

166 Contemporary Topics of Pneumonia

ILDs seen in human CTDs.

the institutional review board.

**Table 5.** Possible immune mechanisms for pulmonary fibrosis.

noticed Raynaud's phenomenon and polyarthralgia, for which she was given low-dose prednisolone with clinical improvement. Two months before the referral, she developed polyarthralgia and myalgia. On physical examination, the patient had no skin lesions but presented with fine crackles audible on inspiration in both lower lung fields. Erythrocyte sedimentation rate (ESR) was 135 mm/hr, and C-reactive protein (CRP) level was 1.2 mg/dL (reference range: 0.0–0.4). Rheumatoid factors (RFs), speckled anti-nuclear factors (ANFs), and anti-histidylt-RNA synthetase (Jo-1) antibodies were positive, while anti-ribonucleoprotein (RNP) and anti-scleroderma-70 (scl-70) antibody assays were negative. Levels of lactate dehydrogenase (LDH, 798 IU/L) (reference range: 109–435), creatine kinase (CK, 559 IU/L) (reference range: 44–140), and myoglobin (120 ng/mL) (reference range: 28–60) were elevated. Muscle strength was nearly normal, but electromyogram showed myogenic patterns in the muscle groups of the upper limb girdle bilaterally. Muscle biopsy revealed lymphocyte infiltration into myofibrils and muscle atrophy, consistent with PM. Arterial blood gas analysis demonstrated a pH of 7.413; partial pressure of carbon dioxide (PCO2 ), 44.6 Torr; partial pressure of oxygen (PO<sup>2</sup> ), 84.3 Torr; and bicarbonate (HCO3 -) concentration, 28.4 mmol/L. Pulmonary function tests revealed a restrictive pattern; vital capacity (VC) was 74.8%, and diffusing capacity (DLco) was 13.44 mL/min/mmHg (67.3%). Analysis of bronchoalveolar lavage fluid (BAL) showed 75% macrophages, 15% neutrophils, and 10% lymphocytes. Human leukocyte antigen (HLA) serotypes were as follows: A2, A26, B15, Cw1, Cw9, DR14, DR8, DR52, DQ7, and DQ6. Chest radiography and chest computed tomography (CT) revealed mild, subpleural, linear, and reticular opacities in posterior and lateral areas of both lungs (**Figure 2a**). The patient underwent video-assisted thoracoscopic (VATS) lung biopsy for histopathologic diagnosis and therapeutic planning. Biopsy specimens from anterior basal segment (S8) and lateral basal segment (S9) of the right lower lobe revealed an early usual IP (UIP) pattern, demonstrating heterogeneous lesions with residual air spaces and early fibrotic changes, surrounded by mild alveolitis with mononuclear cell infiltrations. Based on these clinical and histopathologic findings, the patient was diagnosed with interstitial pneumonia associated with PM. She was subsequently treated with methylprednisolone pulse therapy (1 g/day for 3 days) followed by oral prednisolone (30 mg/day). Over 10 months, the dose of prednisolone was tapered to 12.5 mg/day with excellent control of progression of pulmonary lesions and myositis.

Patient B was a 43-year-old woman, a lifetime nonsmoker, referred to our clinic for arthralgia and myalgia. The patient had developed Raynaud's phenomenon 6 months prior to this presentation. On physical examination, she had swollen fingers, with no skin rash. Fine crackles were heard on inspiration in the lower lung fields. No muscle weakness was apparent, but thorough examination revealed myositis. Laboratory data were as follows: LDH level, 616 IU/L; CK level, 410 IU/L; and CRP level, 0.4 mg/dL. Histological examination of muscle biopsy specimen showed mononuclear cell infiltrates in muscle tissue, consistent with PM. Assays for RF, anti-dsDNA antibodies, anti-Jo-1 antibodies, anti-centromere antibodies, and anti-scl-70 antibodies were negative. ANF (speckled pattern) and anti-ribonucleoprotein (RNP) antibodies were positive. Arterial blood gas analysis was unremarkable. Pulmonary function testing showed a restrictive pattern; VC, 70.6%, and DLco, 14.71 mL/min/mmHg (77.2%). BAL analysis demonstrated 67% macrophages, 11% neutrophils, and 22% lymphocytes. HLA serotypes were as follows: A24, A26, B15, B61, Cw10, DR9, DR53, and DQ9. Chest radiography and CT revealed subpleural and basilar linear and reticular opacities with ground glass attenuation (**Figure 2b**). Histopathological examination of lung specimens from VATS biopsy of superior (S4) and anterior basal (S8) segments of the left lower lobe disclosed a nonspecific IP (NSIP) pattern. Specimens showed mild and homogeneous changes with partial inflammatory thickenings of the alveolar wall, with granulation tissues in alveolar spaces, fibrosis, and inflammatory-cell infiltrations. Clinical and histopathologic findings lead to a diagnosis of IP associated with PM. She was subsequently given pulse therapy with methylprednisolone (1 g/day for 3 days) followed by oral prednisolone (50 mg/day), which was effective in ceasing active myositis and IP. When the dose of prednisolone had been tapered to 30 mg/day after 4 months, respiratory function testing showed that %VC and DLco had

**Figure 2.** (a) Chest radiography, CT, and photomicrograph of patient A. Chest X-ray and HRCT of patient A show subpleural mild linear and reticular opacities in posterolateral lung. Lung biopsy from patient A depicts heterogeneous lesions including residual air spaces and early fibrotic changes with fibroblastic foci, mild alveolitis with thickened alveolar walls, and mononuclear cell infiltrations (hematoxylin and eosin stain, original magnification ×200). (b). Chest radiography, CT, and photomicrograph of patient B. Chest X-ray and HRCT of patient B show thickened interlobular septa with bibasilar and subpleural ground glass opacities. Lung biopsy from patient B depicts mild, homogeneous changes with partial inflammatory thickening of the alveolar wall, with granulation tissues in alveolar spaces, fibrosis,

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Immunohistochemical analysis was performed on lung-infiltrating cells utilizing biopsy specimens. Substantial infiltrations of mononuclear cells were noted in both patient A (UIP pattern) and patient B (NSIP pattern). The mononuclear cells were predominantly CD3+ T cells, accompanied by a subtle infiltration of B cells (CD20+), and a minimal number of monocytes (CD19+). Of infiltrating T cells, CD4+ cells were predominant compared to CD8+ cells in both cases. We then analyzed T cell receptor α-chain variable region (TCR Vα) and TCR β-chain variable region (TCR Vβ) repertoires of T cells infiltrating the lung tissues using an adaptor ligation polymerase chain reaction (PCR)-based microplate hybridization assay [159]. Quantitative assay has been used in many previous studies, and accuracy and reproducibility of this assay have been validated [160, 161]. Briefly, total RNA was extracted with TRIzol

improved to 79.0% and 17.56 mL/min/mmHg (90.7%), respectively.

and inflammatory cell infiltrates (hematoxylin and eosin stain, original magnification ×200).

rate (ESR) was 135 mm/hr, and C-reactive protein (CRP) level was 1.2 mg/dL (reference range: 0.0–0.4). Rheumatoid factors (RFs), speckled anti-nuclear factors (ANFs), and anti-histidylt-RNA synthetase (Jo-1) antibodies were positive, while anti-ribonucleoprotein (RNP) and anti-scleroderma-70 (scl-70) antibody assays were negative. Levels of lactate dehydrogenase (LDH, 798 IU/L) (reference range: 109–435), creatine kinase (CK, 559 IU/L) (reference range: 44–140), and myoglobin (120 ng/mL) (reference range: 28–60) were elevated. Muscle strength was nearly normal, but electromyogram showed myogenic patterns in the muscle groups of the upper limb girdle bilaterally. Muscle biopsy revealed lymphocyte infiltration into myofibrils and muscle atrophy, consistent with PM. Arterial blood gas analysis demonstrated a pH

revealed a restrictive pattern; vital capacity (VC) was 74.8%, and diffusing capacity (DLco) was 13.44 mL/min/mmHg (67.3%). Analysis of bronchoalveolar lavage fluid (BAL) showed 75% macrophages, 15% neutrophils, and 10% lymphocytes. Human leukocyte antigen (HLA) serotypes were as follows: A2, A26, B15, Cw1, Cw9, DR14, DR8, DR52, DQ7, and DQ6. Chest radiography and chest computed tomography (CT) revealed mild, subpleural, linear, and reticular opacities in posterior and lateral areas of both lungs (**Figure 2a**). The patient underwent video-assisted thoracoscopic (VATS) lung biopsy for histopathologic diagnosis and therapeutic planning. Biopsy specimens from anterior basal segment (S8) and lateral basal segment (S9) of the right lower lobe revealed an early usual IP (UIP) pattern, demonstrating heterogeneous lesions with residual air spaces and early fibrotic changes, surrounded by mild alveolitis with mononuclear cell infiltrations. Based on these clinical and histopathologic findings, the patient was diagnosed with interstitial pneumonia associated with PM. She was subsequently treated with methylprednisolone pulse therapy (1 g/day for 3 days) followed by oral prednisolone (30 mg/day). Over 10 months, the dose of prednisolone was tapered to

12.5 mg/day with excellent control of progression of pulmonary lesions and myositis.

Patient B was a 43-year-old woman, a lifetime nonsmoker, referred to our clinic for arthralgia and myalgia. The patient had developed Raynaud's phenomenon 6 months prior to this presentation. On physical examination, she had swollen fingers, with no skin rash. Fine crackles were heard on inspiration in the lower lung fields. No muscle weakness was apparent, but thorough examination revealed myositis. Laboratory data were as follows: LDH level, 616 IU/L; CK level, 410 IU/L; and CRP level, 0.4 mg/dL. Histological examination of muscle biopsy specimen showed mononuclear cell infiltrates in muscle tissue, consistent with PM. Assays for RF, anti-dsDNA antibodies, anti-Jo-1 antibodies, anti-centromere antibodies, and anti-scl-70 antibodies were negative. ANF (speckled pattern) and anti-ribonucleoprotein (RNP) antibodies were positive. Arterial blood gas analysis was unremarkable. Pulmonary function testing showed a restrictive pattern; VC, 70.6%, and DLco, 14.71 mL/min/mmHg (77.2%). BAL analysis demonstrated 67% macrophages, 11% neutrophils, and 22% lymphocytes. HLA serotypes were as follows: A24, A26, B15, B61, Cw10, DR9, DR53, and DQ9. Chest radiography and CT revealed subpleural and basilar linear and reticular opacities with ground glass attenuation (**Figure 2b**). Histopathological examination of lung specimens from VATS biopsy of superior (S4) and anterior basal (S8) segments of the left lower lobe disclosed a nonspecific IP (NSIP) pattern. Specimens showed mild and homogeneous changes with

), 44.6 Torr; partial pressure of oxygen (PO<sup>2</sup>


),

of 7.413; partial pressure of carbon dioxide (PCO2

84.3 Torr; and bicarbonate (HCO3

168 Contemporary Topics of Pneumonia

**Figure 2.** (a) Chest radiography, CT, and photomicrograph of patient A. Chest X-ray and HRCT of patient A show subpleural mild linear and reticular opacities in posterolateral lung. Lung biopsy from patient A depicts heterogeneous lesions including residual air spaces and early fibrotic changes with fibroblastic foci, mild alveolitis with thickened alveolar walls, and mononuclear cell infiltrations (hematoxylin and eosin stain, original magnification ×200). (b). Chest radiography, CT, and photomicrograph of patient B. Chest X-ray and HRCT of patient B show thickened interlobular septa with bibasilar and subpleural ground glass opacities. Lung biopsy from patient B depicts mild, homogeneous changes with partial inflammatory thickening of the alveolar wall, with granulation tissues in alveolar spaces, fibrosis, and inflammatory cell infiltrates (hematoxylin and eosin stain, original magnification ×200).

partial inflammatory thickenings of the alveolar wall, with granulation tissues in alveolar spaces, fibrosis, and inflammatory-cell infiltrations. Clinical and histopathologic findings lead to a diagnosis of IP associated with PM. She was subsequently given pulse therapy with methylprednisolone (1 g/day for 3 days) followed by oral prednisolone (50 mg/day), which was effective in ceasing active myositis and IP. When the dose of prednisolone had been tapered to 30 mg/day after 4 months, respiratory function testing showed that %VC and DLco had improved to 79.0% and 17.56 mL/min/mmHg (90.7%), respectively.

Immunohistochemical analysis was performed on lung-infiltrating cells utilizing biopsy specimens. Substantial infiltrations of mononuclear cells were noted in both patient A (UIP pattern) and patient B (NSIP pattern). The mononuclear cells were predominantly CD3+ T cells, accompanied by a subtle infiltration of B cells (CD20+), and a minimal number of monocytes (CD19+). Of infiltrating T cells, CD4+ cells were predominant compared to CD8+ cells in both cases. We then analyzed T cell receptor α-chain variable region (TCR Vα) and TCR β-chain variable region (TCR Vβ) repertoires of T cells infiltrating the lung tissues using an adaptor ligation polymerase chain reaction (PCR)-based microplate hybridization assay [159]. Quantitative assay has been used in many previous studies, and accuracy and reproducibility of this assay have been validated [160, 161]. Briefly, total RNA was extracted with TRIzol Reagent (Invitrogen, USA) from lung tissues obtained by VATS biopsy. Complementary DNA (cDNA) was synthesized with reverse transcriptase (Superscript II) and BSL-18E primer adaptor. Universal adaptor primers were ligated to the cDNA, and PCR was performed with the universal primer and primers specific for TCR Cα or Cβ chains. PCR products were biotinylated by amplification with 5′-biotin primer and the universal primer. The biotinylated PCR products were hybridized on microplate wells immobilized with various oligo probes specific for TCR Vα or Vβ region sequences. Finally, the amount of biotinylated product bound to each probe was measured by quantitative enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase streptavidin. The results of quantitative analysis of TCR Vα or TCR Vβ usage in patient A and B are shown in **Figure 3a** and **b**, respectively. The open column and solid column indicate the frequencies of TCR Vα/Vβ repertoires in the lung tissue and the peripheral blood lymphocytes (PBLs), respectively. We found that the usage of repertoires of TCR Vα/Vβ in the lung differed from those in PBL, with certain TCR V gene families detected more frequently from lung tissue. In patient A, TCR repertoires of VA2.1, VA8.1, VB4.1, and VB24.1 were predominantly expressed in the lung compared to PBL, while TCR V repertoires VA23.1 and VB13.1 were more frequently expressed in lung tissue from patient B. As expected, no TCR signals were detected on normal lung tissue obtained from patients without IP using the same method (data not shown).

#### *6.2.2. Discussion*

IP associated with PM/DM is recognized as a critical complication due to its association with poor disease prognosis. In this study, we investigated T cells infiltrates by analyzing the TCR repertoire usage in lung tissue in two patients with PM-associated early IP. Both patients showed marked lymphocytes aggregates, predominantly CD3+ T cells, at IP lesion sites. In addition, the analysis of TCR Vα/Vβ repertoire usage indicated a modest accumulation of T cells expressing selected TCR V-gene segments, which differed distinctly from those of PBL. These findings strongly suggest pathogenic involvement of organ-specific oligoclonal T cell accumulation in development of PM-associated IP. Since TCR diversifies with disease progression due to the phenomenon of "determinant spreading" in which autoreactive T cell responses, initiated by a single antigenic epitope, evolve into multiepitopic responses, we considered it important to perform TCR analysis from lung tissue in the earliest stage of IP [162]. Regarding differences in predominant TCR V gene usage between the two cases, we suspect this is due to HLA differences. A previous study on lung TCR repertoire in patients with PM by Englund et al. reported selective TCR V gene usage, characterized by a panel of TCR-specific monoclonal antibodies on flow cytometry [163]. However, that study used BAL fluid rather than lung tissue in which infiltrating T cells may be more directly involved in the disease process. Although we also tried to analyze TCR repertoire from BAL fluid, the data were inconclusive due to lack of TCR signals along with background noise from RNA debris. To our knowledge, this is the first robust demonstration of the presence of selective TCR V gene usage and its differential expression in lung tissues of patients with PM using both lung biopsy tissue from early IP and PBL. Because our study involved only two cases, these findings should be confirmed in a larger study. However, we believe these findings strongly suggest that T cells which are recruited into the lung may be exposed to autoantigens, selectively expanding by antigen-driven responses. Further studies are needed to identify T cell epitopes

**Figure 3.** (a) Quantitative analysis of TCR repertoires: TCR Vα gene usage (top) and TCR Vβ gene usage (bottom) in patient A. Solid and open bars indicate frequencies of TCR Vα/Vβ repertoires in PBL and lung tissue, respectively. VA2.1 and VA8.1 and VB4.1 and VB24.1 repertoires predominate in lung tissue compared with PBL. (b). Quantitative analysis of TCR repertoires: TCR Vα gene usage (top) and TCR Vβ gene usage (bottom) in patient B. Solid and open bars indicate frequencies of TCR Vα/Vβ repertoires in PBL and lung tissue, respectively. VA23.1 and VB13.1 repertoires are more

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frequent in lung tissue compared with PBL.

Reagent (Invitrogen, USA) from lung tissues obtained by VATS biopsy. Complementary DNA (cDNA) was synthesized with reverse transcriptase (Superscript II) and BSL-18E primer adaptor. Universal adaptor primers were ligated to the cDNA, and PCR was performed with the universal primer and primers specific for TCR Cα or Cβ chains. PCR products were biotinylated by amplification with 5′-biotin primer and the universal primer. The biotinylated PCR products were hybridized on microplate wells immobilized with various oligo probes specific for TCR Vα or Vβ region sequences. Finally, the amount of biotinylated product bound to each probe was measured by quantitative enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase streptavidin. The results of quantitative analysis of TCR Vα or TCR Vβ usage in patient A and B are shown in **Figure 3a** and **b**, respectively. The open column and solid column indicate the frequencies of TCR Vα/Vβ repertoires in the lung tissue and the peripheral blood lymphocytes (PBLs), respectively. We found that the usage of repertoires of TCR Vα/Vβ in the lung differed from those in PBL, with certain TCR V gene families detected more frequently from lung tissue. In patient A, TCR repertoires of VA2.1, VA8.1, VB4.1, and VB24.1 were predominantly expressed in the lung compared to PBL, while TCR V repertoires VA23.1 and VB13.1 were more frequently expressed in lung tissue from patient B. As expected, no TCR signals were detected on normal lung tissue obtained from patients without

IP associated with PM/DM is recognized as a critical complication due to its association with poor disease prognosis. In this study, we investigated T cells infiltrates by analyzing the TCR repertoire usage in lung tissue in two patients with PM-associated early IP. Both patients showed marked lymphocytes aggregates, predominantly CD3+ T cells, at IP lesion sites. In addition, the analysis of TCR Vα/Vβ repertoire usage indicated a modest accumulation of T cells expressing selected TCR V-gene segments, which differed distinctly from those of PBL. These findings strongly suggest pathogenic involvement of organ-specific oligoclonal T cell accumulation in development of PM-associated IP. Since TCR diversifies with disease progression due to the phenomenon of "determinant spreading" in which autoreactive T cell responses, initiated by a single antigenic epitope, evolve into multiepitopic responses, we considered it important to perform TCR analysis from lung tissue in the earliest stage of IP [162]. Regarding differences in predominant TCR V gene usage between the two cases, we suspect this is due to HLA differences. A previous study on lung TCR repertoire in patients with PM by Englund et al. reported selective TCR V gene usage, characterized by a panel of TCR-specific monoclonal antibodies on flow cytometry [163]. However, that study used BAL fluid rather than lung tissue in which infiltrating T cells may be more directly involved in the disease process. Although we also tried to analyze TCR repertoire from BAL fluid, the data were inconclusive due to lack of TCR signals along with background noise from RNA debris. To our knowledge, this is the first robust demonstration of the presence of selective TCR V gene usage and its differential expression in lung tissues of patients with PM using both lung biopsy tissue from early IP and PBL. Because our study involved only two cases, these findings should be confirmed in a larger study. However, we believe these findings strongly suggest that T cells which are recruited into the lung may be exposed to autoantigens, selectively expanding by antigen-driven responses. Further studies are needed to identify T cell epitopes

IP using the same method (data not shown).

*6.2.2. Discussion*

170 Contemporary Topics of Pneumonia

**Figure 3.** (a) Quantitative analysis of TCR repertoires: TCR Vα gene usage (top) and TCR Vβ gene usage (bottom) in patient A. Solid and open bars indicate frequencies of TCR Vα/Vβ repertoires in PBL and lung tissue, respectively. VA2.1 and VA8.1 and VB4.1 and VB24.1 repertoires predominate in lung tissue compared with PBL. (b). Quantitative analysis of TCR repertoires: TCR Vα gene usage (top) and TCR Vβ gene usage (bottom) in patient B. Solid and open bars indicate frequencies of TCR Vα/Vβ repertoires in PBL and lung tissue, respectively. VA23.1 and VB13.1 repertoires are more frequent in lung tissue compared with PBL.

of the pathogenic antigens, which may potentially lead to the development of antigen-specific, molecular-targeted therapies, such as the induction of anergy by peptide analogues similar in structure to culprit antigens [164, 165].

recent systematic review and meta-analysis of RCTs and observational prospective cohort studies failed to validate any clinically significant improvement in pulmonary function in SSc

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In PM/DM-associated ILD, high-dose steroid is often the first-line drug, although no definite therapeutic recommendation for the disease has been established yet. The other drugs most frequently used are AZA, MMF, hydroxychloroquine, MTX, CYC, and calcineurin inhibitors, e.g., cyclosporine A (CSA) and tacrolimus (TAC). Rituximab, anti-CD20 monoclonal antibody therapy, has lately emerged as a promising remedy of biologics in patients who have failed conventional immunosuppression treatments [171, 172]. Among a variety of immunosuppressants, the efficacy of calcineurin inhibitors for the treatment of PM/DM-associated ILD should be highlighted. CSA, which inhibits T cell proliferation and T cell-mediated cytokine productions at the transcriptional level, has begun to be used for PM/DM-ILD since the 1980s [173–175]. In 1998, the first nation-wide survey for the treatment with CSA in IP associated with CTDs was conducted in Japan, and the efficacy of a combination therapy with CSA and corticosteroids in PM/DM associated IP was indicated [176]. A number of retrospective and open-label studies have supported the benefit of CSA for the treatment of ILD with PM/DM [177–181]. Takada et al. published a retrospective multicenter study of 38 cases with acute ILD with PM/ DM, whereas it was shown that the combination therapy with CSA and corticosteroids started from the early phase of ILD is superior to corticosteroid monotherapy [178]. Today, calcineurin inhibitors are widely used especially in Japan as both an induction and maintenance therapy for PM/DM-ILD, generally resulting in favorable prognostic outcomes. The appropriate serum concentration of CSA to ensure a maximal effect as well as to avoid toxicity in patients with PM/DM-ILD should reach approximately 150 ng/mL and1000 ng/mL, at trough and at 2 hours after administration, respectively [179]. Another calcineurin inhibitor, TAC, which is a 100 fold potent T cell inhibitor compared to CSA, was also introduced into the treatment for PM/ DM-ILD, and its efficacy and tolerability have been demonstrated in retrospective studies and case series since the report by Oddis et al. in 1999 [182–185]. Ochi, et al. described a superior effect of TAC used in two myositis patients with progressive ILD who failed CYC and corticosteroid treatment but successfully recovered with TAC, showing significant improvement in symptoms and radiologic changes [183]. The appropriate tacrolimus trough level for the treatment of ILD in PM/DM patients have not been established by clinical trials, but it is usually set

as 5–20 ng/mL on the basis of data from renal and bone marrow transplantation [186].

Given the treatment-effect heterogeneity of the lung disease observed in PM/DM, it is important to prepare novel therapeutics for the challenging cases of ILD which are refractory to conventional formulas. Recently, Suda et al. reported the effectiveness of multitarget therapy for the ILD in two cases of anti-MDA5 antibody-positive DM which is known to be associated with progressive ILD and sometimes has a lethal outcome despite strong immunosuppressive therapy including CYC [187]. They used TAC and mizoribine (MZR, an inosine monophosphate dehydrogenase inhibitor) in combination with corticosteroids. MZR is a nucleoside of the imidazole class, with the same mechanism as MMF: selective inhibition of lymphocyte proliferation by blocking inosine monophosphate dehydrogenase [188]. The safety and steroid-sparing effects of MZR have been shown in various CTDs, and the efficacy of multitarget therapy using TAC and MZR was reported for systemic lupus erythematosus [189, 190].

patients treated with CYC [169, 170].

Thus, as a result, the T cell receptor (TCR) repertoire study combined with histological analysis demonstrated substantial CD3+ T cell lung infiltrates with specific oligoclonal TCR usage that differed from those in PBL, suggesting a pivotal role for T cells in the pathogenesis of PM-associated IP via antigen-driven immune mechanisms.
