Abstract
Acute respiratory distress syndrome (ARDS) is a serious complication of systemic inflammatory response syndrome, and diffuse alveolar damage (DAD) is a histological manifestation of ARDS. Endothelial cell injury is mainly responsible for ARDS. Many neutrophils and macrophages/monocytes, which are inflammatory cells that play a role in innate immunity, infiltrate the lung tissue in DAD. In recent years, it has become clear that CD8 plays an important role not only in the acquired immune system, but also in the innate immune system. Non-antigen-activated bystander CD8 + T cells express the unique granzyme B (GrB) + /CD25-/programmed cell death-1 (PD-1)-phenotype. The involvement of bystander CD8 + T cells in lung tissue in DAD is an unexplored field. This study aimed to determine whether bystander CD8 is involved in DAD. Twenty-three consecutive autopsy specimens were retrieved from patients with DAD, and the phenotypes of infiltrating lymphocytes in the DAD lesions were evaluated using immunohistochemistry. In most cases, the number of CD8 + T cells was higher than that of CD4 + T cells, and many GrB + cells were also observed. However, the number of CD25 + and PD-1 + cells was low. We conclude that bystander CD8 + T cells may be involved in cell injury during the development of DAD.
Keywords: Bystander, CD8 T cells, Diffuse alveolar damage, Innate immunity
Introduction
Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterised by progressive respiratory insufficiency caused by diffuse alveolar damage (DAD) resulting from sepsis or severe trauma. Damage to the endothelium and alveolar epithelial cells is a key event in the development of DAD. Neutrophils and macrophages/monocytes are believed to play important roles in the pathogenesis of DAD. In the acute phase of DAD, neutrophils and macrophages/monocytes infiltrate the interstitium and alveoli [1]. In ARDS, circulating cytokines may directly activate the pulmonary endothelium, which leads to endothelial injury and/or activates endothelial cells to express increased levels of adhesion molecules [1]. Neutrophils then adhere to the activated endothelium and migrate into the interstitium and alveoli, causing more endothelial injury [1]. This increases the vascular permeability and inhibits surfactant function, thereby decreasing alveolar expansion. Histologically, oedema and hyaline membrane formation are observed in the acute phase, and type II pneumocyte proliferation and granulation tissue in the alveolar walls and spaces are observed in the organising phase [1].
CD8 is a cytotoxic lymphocyte that is activated in an antigen-dependent manner and plays a role in acquired immunity. In recent years, however, the existence of bystander CD8 + T cells, which is activated by cytokines in an antigen-independent manner and causes cell injury, has been identified [2]. It has become clear that CD8 plays an important role not only in the acquired immune system as conventionally believed, but also in the innate immune system. For example, bystander CD8 + T cells are the major infiltrating cytotoxic lymphocyte in acute viral hepatitis and influenza virus pneumonia [3, 4].
Bystander CD8 + T cells exhibit a unique phenotype. They express granzyme B (GrB) but do not upregulate CD25 or programmed cell death-1 (PD-1) [5]. CD25 and PD-1 can be induced by antigen-dependent signals through T cell receptors or CD3 [5]. The predominant infiltration of bystander CD8 + T cells with the GrB + /CD25-/PD-1- phenotype has been reported in other pathological specimens, such as cancer tissue after cytokine immunotherapy [5], lung tissue after influenza virus infection [3], and the intima of polyarteritis nodosa-type vasculitis [6]. Under each condition, antigen-independent activated bystander CD8 + T cells were the predominant infiltrating lymphocytes. However, the involvement of bystander CD8 + T cells in DAD remains unexplored.
This study aimed to determine whether bystander CD8 + T cells are involved in cell injury in DAD. In this study, we examined the extent of bystander CD8 + T cell infiltration in the alveolar septa via immunohistochemistry analysis of lung tissue samples from autopsied patients who died from sepsis or other conditions leading to ARDS/DAD.
Materials and methods
Cases
This study was approved by the ethics committee of the Shinshu University School of Medicine, Japan (No. 5146) and the ethics committee of Marunouchi Hospital, Matsumoto, Japan (No.22–24). Consecutive histopathological slides of autopsy specimens from 23 cases of DAD were retrieved from the pathology files at the Department of Laboratory Medicine, Shinshu University Hospital and affiliated hospitals, Matsumoto, Japan, between 2006 and 2022. Patients with underlying lung diseases, such as chronic interstitial pneumonia, viral pneumonia, infiltration of lymphoma or leukaemia cells, and primary or metastatic lung cancer, were excluded. According to clinical records and autopsy reports, the causes of ARDS and DAD were sepsis (n = 18), severe hepatic failure (n = 2), cardiogenic shock (n = 2), and haemorrhagic shock (n = 1). Among the 18 sepsis cases, the pathogenic organism could be identified in 10 cases, with Staphylococcus spp. in 4 cases, Klebsiella spp. in 2 cases, Escherichia coli in 2 cases, Enterococcus feacium in one case, and Corynebacterium striatum in one case. In the remaining 8 cases of sepsis, the pathogenic organism was unknown. The mean age of the patients was 69.1 years (range 38–92), and the male-to-female ratio was 1.88:1.
Histopathological evaluation and selection of tissue samples
Two experienced pathologists (M.O. and M.K.) reviewed the archived haematoxylin and eosin (HE)-stained slides to confirm the findings of DAD. One representative paraffin block containing the alveolar region, reflecting the acute phase of DAD (Fig. 1a) was selected for immunohistochemical analysis in each case. Most cases showed acute phase DAD lesions as well as organizing phase DAD lesions (Fig. 1b). In some cases, lesions only showing the acute phase of DAD changes were found, with no appreciable lesions showing the organizing phase of DAD changes. At the same time, areas that were thought to be more affected by smoking, such as emphysematous areas, were excluded. All the tissue samples were fixed in formalin and embedded in paraffin. Serial tissue Sects. (3 μm-thick) were prepared for HE staining and immunohistochemical analysis.
Fig. 1.
Histopathology of autopsy specimens of the acute phase (a) and the organizing phase (b) of diffuse alveolar damage (DAD). Haematoxylin and eosin staining. In the acute phase, oedema and hyaline membrane formation are observed (a). In the organizing phase, granulation tissue forms in the alveolar walls and spaces (b). Original magnification × 100
Immunohistochemistry
Formalin pigment was removed using alcohol solutions of potassium hydroxide for 1 h at room temperature. The sections were incubated in 3% H2O2 for 10 min at room temperature to quench endogenous peroxidase activity. Antigen retrieval was performed by heating the sections in 10 mM EDTA buffer (pH 8.0) in a microwave oven at 600 W for 30 min. Infiltrating lymphocytes in DAD were immunophenotyped by incubating the membranes with the following mouse monoclonal antibodies overnight at 4 °C: anti-CD3 (clone LN10; Novocastra, Newcastle, UK), anti-CD4 (clone 1F6; Novocastra), anti-CD8 (clone 1A5; Novocastra), anti-CD20 (clone L26; Dako, Glostrup, Denmark), anti-CD25 (clone 4C9; Leica, Newcastle, UK), anti-Granzyme B (GrB) (clone GrB-7; SanBio, Tokyo, Japan), and anti-programmed cell death-1 (PD-1) (clone ab137132; Abcam, Uden, The Netherlands). For CD3, CD8, and CD25, subsequent signal development was performed using the immunoenzyme polymer method (Novolink Polymer Detection System; Leica, Milton Keynes, UK) with 3,3′-diaminobenzidine (DAB) kits (Histofine DAB Kit, Nichirei, Tokyo, Japan). For CD20 and PD-1, subsequent signal development was performed using the immunoenzyme polymer method (Histofine Simple Stain MAX PO Multi; Nichirei) with the chromogen DAB. For CD4 and GrB, signal development was performed using the immunoenzyme polymer method (Novolink Polymer Detection System; Leica) with the chromogen DAB. Double immunostaining with anti-CD8 and anti-GrB antibodies was also performed. Briefly, immunostaining for GrB was performed as described above, except that DAB with NiCl2 was used as the chromogen. After blocking the remaining peroxidase activity with 0.3% H2O2 in methanol for 15 min, immunostaining for CD8 was performed as described above. Pulmonary hilar lymph nodes of the autopsy specimens were used as positive controls for immunohistochemical staining.
Evaluation of infiltrating inflammatory cells
The number of infiltrating lymphocytes was evaluated using immunohistochemistry. A single pathologist (M.K.) selected three areas (each 0.5 mm2) showing the acute phase of DAD changes (Fig. 1a) and the organizing phase of DAD changes (Fig. 1b) in HE staining, while two other pathologists validated the areas (M.O. and A.T.). Next, areas corresponding to the same three areas selected in the above HE-stained specimens were marked in each immunostained specimen, and the number of immunopositive cells infiltrating the areas was counted and summed. HE-stained-specimens and immunostained specimen were prepared in serial tissue sections. Immunopositive cells were counted manually by three experienced pathologists (M.O., A.T., and M.K.) independently, and the average value was calculated.
Statistical analysis
Statistical analysis was performed using the Mann-Whitney test. A p value < 0.05 was considered significant.
Results
Representative histopathological findings in the acute phase and organizing phase of DAD lesions are shown in Figs. 2 and 3, respectively. The average number of infiltrating immunopositive cells in each case of the acute phase and organizing phase of DAD lesions is listed in Tables 1 and 2, respectively. Furthermore, the mean and median values of the number of immunopositive cells in each of the 23 cases of the acute and organinzing phases of DAD lesions are shown as boxplots (Fig. 4).
Fig. 2.
Histopathology and immunostaining of autopsy specimens of the acute phase of diffuse alveolar damage (DAD). Haematoxylin and eosin staining (a), immunostaining of CD20 (b), CD3 (c), CD4 (d), CD8 (e), granzyme B (GrB) (f), CD25 (g), and programmed cell death-1 (PD-1) (h), and double immunostaining of CD8 and GrB (i). CD20 + B cells are almost absent (a). Many CD3 + , CD4 + , and/or CD8 + T cells infiltrated the alveolar septa in DAD (b-e). Many GrB + cells are also observed (f). The number of CD25 + or PD-1 + cells is low (g, h). Many CD8 (brown)/GrB (black) double-positive cells are observed (i). a-h; Original magnification × 400; i: Original magnification × 1000 oil
Fig. 3.
Histopathology and immunostaining of autopsy specimens of the organizing phase of diffuse alveolar damage (DAD). Haematoxylin and eosin staining (a), immunostaining for CD20 (b), CD3 (c), CD4 (d), CD8 (e), granzyme B (GrB) (f), CD25 (g), and programmed cell death-1 (PD-1) (h), and double immunostaining for CD8 and GrB (i). CD20 + B cells are almost absent (a). Many CD3 + , CD4 + , and/or CD8 + T cells infiltrated the alveolar septa and the organinzing lesions in DAD (b-e). Many GrB + cells are also observed (f). Few CD25 + or PD-1 + cells are observed (g, h). Many CD8 (brown)/GrB (black) double-positive cells are observed (i). a-h; Original magnification × 400; i: Original magnification × 1000 oil
Table 1.
Summary of clinical data and number of immunopositive cells in 23 cases with early-stage ARDS/DAD
| Case no | Cause of ARDS/DAD | Age (years) | Sex | Number of immuno-positive cells | Ratio of lymphocytes | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CD20 | CD3 | CD4 | CD8 | GrB | CD25 | PD-1 | CD3/CD20 | CD8/CD4 | ||||
| 1 | Severe hepatic failure | 38 | M | 7.3 | 122.3 | 43.0 | 73.3 | 35.7 | 10.0 | 12.0 | 16.8 | 1.7 |
| 2 | Severe hepatic failure | 44 | M | 13.3 | 72.3 | 18.7 | 50.3 | 45.7 | 1.7 | 19.3 | 5.4 | 2.7 |
| 3 | Sepsis; Corynebacterium striatum | 74 | M | 18.3 | 294.7 | 47.0 | 189.3 | 126.0 | 6.7 | 31.3 | 16.1 | 4.0 |
| 4 | Sepsis* | 81 | M | 4.3 | 82.0 | 27.0 | 44.0 | 29.3 | 4.3 | 6.3 | 19.0 | 1.6 |
| 5 | Sepsis* | 80 | F | 0.3 | 65.7 | 24.7 | 62.7 | 17.7 | 1.3 | 6.7 | 197.0 | 2.5 |
| 6 | Sepsis* | 78 | M | 2.3 | 124.7 | 43.0 | 82.0 | 16.0 | 8.3 | 6.0 | 54.2 | 1.9 |
| 7 | Sepsis; Klebsiella pneumoniae | 73 | F | 18.3 | 142.7 | 42.3 | 123.0 | 103.7 | 4.0 | 15.0 | 7.8 | 2.9 |
| 8 | Sepsis; Escherichia coli | 62 | M | 3.3 | 98.0 | 39.7 | 60.3 | 13.7 | 4.7 | 8.7 | 29.7 | 1.5 |
| 9 | Sepsis; Escherichia coli | 67 | M | 4.7 | 117.7 | 43.3 | 85.3 | 21.0 | 5.7 | 7.7 | 25.0 | 2.0 |
| 10 | Sepsis* | 44 | F | 11.3 | 87.3 | 45.0 | 48.0 | 10.3 | 5.0 | 5.3 | 7.7 | 1.0 |
| 11 | Haemorrhagic shock | 71 | M | 15.7 | 113.0 | 50.7 | 85.3 | 55.7 | 6.3 | 4.0 | 7.2 | 1.7 |
| 12 | Sepsis* | 62 | M | 4.7 | 88.7 | 24.7 | 88.7 | 19.0 | 1.7 | 14.0 | 18.9 | 3.6 |
| 13 | Sepsis; Staphylococcus aureus | 38 | M | 2.0 | 169.0 | 38.3 | 114.3 | 39.3 | 2.0 | 13.0 | 84.5 | 3.0 |
| 14 | Sepsis; Staphylococcus aureus | 86 | M | 3.0 | 79.7 | 15.7 | 52.0 | 30.7 | 4.3 | 6.3 | 26.6 | 3.3 |
| 15 | Cardiogenic shock | 80 | F | 1.3 | 51.3 | 20.0 | 26.7 | 17.7 | 1.3 | 1.0 | 39.5 | 1.3 |
| 16 | Sepsis; Staphylococcus aureus | 68 | M | 5.0 | 404.3 | 142.0 | 212.7 | 110.3 | 18.3 | 11.0 | 80.9 | 1.5 |
| 17 | Sepsis* | 82 | F | 7.3 | 162.0 | 52.0 | 106.7 | 73.7 | 5.7 | 7.3 | 22.2 | 2.1 |
| 18 | Sepsis* | 80 | M | 6.3 | 70.3 | 21.7 | 46.7 | 38.3 | 2.3 | 5.0 | 11.2 | 2.2 |
| 19 | Cardiogenic shock | 92 | F | 9.7 | 202.0 | 78.0 | 127.7 | 92.0 | 7.0 | 9.7 | 20.8 | 1.6 |
| 20 | Sepsis; Enterococcus feacium | 72 | M | 12.7 | 452.0 | 143.0 | 305.3 | 276.7 | 49.3 | 15.0 | 35.6 | 2.1 |
| 21 | Sepsis* | 80 | M | 2.3 | 76.7 | 19.7 | 46.3 | 35.0 | 5.0 | 5.0 | 33.3 | 2.4 |
| 22 | Sepsis; Klebsiella oxytoca | 60 | F | 6.0 | 72.7 | 33.3 | 54.3 | 50.7 | 7.0 | 4.0 | 12.1 | 1.6 |
| 23 | Sepsis; Staphylococcus simulans | 78 | F | 11.0 | 176.0 | 36.7 | 142.7 | 78.7 | 3.3 | 4.0 | 16.0 | 3.9 |
ARDS acute respiratory distress syndrome, DAD diffuse alveolar damage, GrB granzyme B, PD-1 programmed cell death 1
*The pathogenic organism was unknown
Table 2.
Summary of clinical data and number of immunopositive cells in 23 cases with organizing-stage ARDS/DAD
| Case no | Cause of ARDS/DAD | Age (years) | Sex | Number of immuno-positive cells | Ratio of lymphocytes | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CD20 | CD3 | CD4 | CD8 | GrB | CD25 | PD-1 | CD3/CD20 | CD8/CD4 | ||||
| 1 | Severe hepatic failure | 38 | M | 9.3 | 212.7 | 87.0 | 103.3 | 45.0 | 11.0 | 19.7 | 22.9 | 1.2 |
| 2 | Severe hepatic failure | 44 | M | ※No evaluable organizing lesion | ||||||||
| 3 | Sepsis; Corynebacterium striatum | 74 | M | 26.0 | 513.7 | 222.3 | 283.3 | 97.7 | 8.7 | 72.0 | 19.7 | 1.3 |
| 4 | Sepsis* | 81 | M | 9.0 | 173.7 | 65.0 | 79.7 | 38.3 | 10.0 | 20.7 | 19.3 | 1.2 |
| 5 | Sepsis* | 80 | F | 3.3 | 110.3 | 47.7 | 75.3 | 21.3 | 15.0 | 12.3 | 33.4 | 1.6 |
| 6 | Sepsis* | 78 | M | ※No evaluable organizing lesion | ||||||||
| 7 | Sepsis; Klebsiella pneumoniae | 73 | F | 20.7 | 196.7 | 70.0 | 129.7 | 79.7 | 3.0 | 23.3 | 9.5 | 1.9 |
| 8 | Sepsis; Escherichia coli | 62 | M | 8.3 | 181.7 | 70.7 | 106.3 | 19.7 | 13.0 | 32.3 | 21.9 | 1.5 |
| 9 | Sepsis; Escherichia coli | 67 | M | 3.7 | 138.0 | 66.3 | 80.7 | 23.3 | 11.0 | 11.7 | 37.3 | 1.2 |
| 10 | Sepsis* | 44 | F | ※No evaluable organizing lesion | ||||||||
| 11 | Haemorrhagic shock | 71 | M | 17.0 | 148.0 | 78.0 | 73.0 | 46.7 | 11.7 | 13.0 | 8.7 | 0.9 |
| 12 | Sepsis* | 62 | M | 7.0 | 113.3 | 46.3 | 87.3 | 28.7 | 9.3 | 27.0 | 16.2 | 1.9 |
| 13 | Sepsis; Staphylococcus aureus | 38 | M | 9.0 | 261.0 | 79.7 | 180.0 | 95.3 | 10.3 | 37.0 | 29.0 | 2.3 |
| 14 | Sepsis; Staphylococcus aureus | 86 | M | 10.3 | 127.3 | 34.7 | 97.0 | 41.7 | 10.7 | 8.3 | 12.4 | 2.8 |
| 15 | Cardiogenic shock | 80 | F | 3.0 | 115.0 | 18.0 | 76.7 | 38.3 | 2.7 | 5.0 | 38.3 | 4.3 |
| 16 | Sepsis; Staphylococcus aureus | 68 | M | ※No evaluable organizing lesion | ||||||||
| 17 | Sepsis* | 82 | F | ※No evaluable organizing lesion | ||||||||
| 18 | Sepsis* | 80 | M | ※No evaluable organizing lesion | ||||||||
| 19 | Cardiogenic shock | 92 | F | ※No evaluable organizing lesion | ||||||||
| 20 | Sepsis; Enterococcus feacium | 72 | M | 30.0 | 539.3 | 202.0 | 331.3 | 282.3 | 77.3 | 29.0 | 18.0 | 1.6 |
| 21 | Sepsis* | 80 | M | 3.0 | 155.7 | 54.0 | 80.3 | 45.7 | 7.0 | 7.3 | 51.9 | 1.5 |
| 22 | Sepsis; Klebsiella oxytoca | 60 | F | ※No evaluable organizing lesion | ||||||||
| 23 | Sepsis; Staphylococcus simulans | 78 | F | 55.7 | 362.7 | 81.0 | 286.3 | 88.0 | 14.0 | 10.7 | 6.5 | 3.5 |
ARDS acute respiratory distress syndrome, DAD diffuse alveolar damage, GrB granzyme B, PD-1 programmed cell death 1
*The pathogenic organism was unknown
Fig. 4.
Boxplots showing the mean and median values of the number of immunopositive cells in each of the 23 cases with both the acute and organinzing phases of DAD. a The mean and median values of the number of CD20 + cells are lower than that of CD3 + cells; in the organizing phase, the number of CD3 + cells is significantly increased compared to those in the acute phase (p < 0.01), while the differences in the number of CD20 + cellsare not statistically significant. b The number of CD4 + cells is lower than that of CD8 + cells; in the organizing phase, the number of CD4 + cells is significantly increased compared to that in the acute phase (p < 0.01), while the differences in the number of CD8 + cells are not statistically significant. c The number of CD25 + or PD-1 + cells are lower than that of GrB + cells; in the organizing phase, the number of CD25 + and PD-1 + cells is significantly increased compared to that the acute phase (p < 0.01), but there is no statistically significant difference in the number of GrB + cells. The P-values were obtained using Mann–Whitney tests
In the acute phase of DAD lesions, CD20 + B cells were almost absent (Fig. 2a). Many CD3 + , CD4 + , and/or CD8 + T cells infiltrated the alveolar septa in DAD (Fig. 2b-e). CD3 + T cells predominated over CD20 + B cells and CD8 + T cells predominated over CD4 + T cells. Many GrB + cells were also identified (Fig. 2f). The number of CD25 + or PD-1 + cells was lower than that of CD3 + , CD4 + , CD8 + , or GrB + cells (Fig. 2g and h). Double immunostaining for CD8 and GrB did not provide sufficient staining for accurate counting; however, many CD8 (brown)/GrB (black) double-positive cells were observed (Fig. 2i).
In the organizing phase of DAD lesions, a few CD20 + B cells were found (Fig. 3a). Many CD3 + , CD4 + , and/or CD8 + T cells also infiltrated the alveolar septa and organinzing lesions (Fig. 3b-e). Many GrB + cells were also identified (Fig. 3f). Although the number of CD25 + or PD-1 + cells was higher than that in the acute phase, it was lower than that of CD3 + , CD4 + , CD8 + or GrB + cells (Fig. 3g and h). Double immunostaining for CD8 and GrB also revealed many CD8 (brown)/ GrB (black) double positive cells (Fig. 3i).
The box prot graph indicated that the mean and median values of the number of CD20 + cells were lower than that of CD3 + cells in both the acute and organinzing phases of DAD (Fig. 4a). The number of CD4 + cells was lower than that of CD8 + cells in both the acute and organinzing phases of DAD (Fig. 4b). In the organizing phase, the number of CD3 + cells and CD4 + cells was significantly increased compared to that in the acute phase (p < 0.01), while the differences in the number of CD20 + cells and CD8 + cells were not statistically significant. The number of CD25 + or PD-1 + cells was lower than that of GrB + cells in both the acute and organinzing phases of DAD (Fig. 4c). In the organizing phase, the number of CD25 + and PD-1 + cells was significantly increased compared to that in the acute phase (p < 0.01), but there was no statistically significant difference in the number of GrB + cells.
Discussion
Bystander CD8 + T cells may have the advantage of faster responses than antigen–antibody responses. Therefore, bystander CD8 + T cells may be useful for antitumour effects or protection against infection. However, its potential role as an aggravating factor in acute inflammatory diseases cannot be ruled out. In this study, it was found that many CD8 + T cells, GrB + cells, and CD8 + /GrB + cells infiltrated during the acute phase of DAD, while the number of CD25 + andI PD-1 + cells was low. Although double immunostaining for GrB/CD25 and GrB/PD-1 was attempted, the fixation conditions were poor because of the autopsy material, and the staining was not successful. However, since there were very few positive cells for CD25 and PD-1 in single staining, it was speculated that antigen-specific activated CD8 + T cells with the GrB( +)/CD25( +)/PD-1( +) trait would be a minority. Previous studies have also reported infiltration of many CD4 + and CD8 + T cells in both viral and non-viral acute lung injury; however, CD25 + cells were scarce [7]. Infiltration of bystander CD8 + T cells in the lungs during the acute phase of DAD and their cytotoxicity may form part of the pathogenesis of DAD.
In DAD, infiltration of a large number of lymphocytes, neutrophils, and macrophages/monocytes has been reported [8, 9]. In terms of the phenotype of infiltrating lymphocytes, CD3 + T cells predominated over CD20 + B cells, suggesting that cytotoxic immune mechanisms mediated by T cells are involved in the pathogenesis of the disease [8, 9]. In addition, the ratio of infiltrating CD4 + T cells to CD8 + T cells was slightly CD8-dominant [7–9], which is consistent with the result observed in the present study. In animal studies, CD8 has been reported to be involved in ARDS pathogenesis [10, 11]. Cytotoxicity due to CD8 + T cells along with neutrophil cytotoxicity may be involved in the development of DAD.
In addition, the present study suggests that a majority of CD8 + T cells appear to be activated in an antigen-independent manner. If non-pathological antigen–antibody reactions occur in vivo, inflammation will not occur and will not take the form of acute inflammation that forms "lesions". Therefore, the majority of inflammatory cells infiltrating in acute inflammatory "lesions" may not be acquired immune system cells responsible for antigen–antibody reactions but innate immune system cells, such as neutrophils, macrophages/monocytes, and bystander CD8 + T cells. In recent years, attention has been given not only on the conventional flow of "innate immunity → acquired immunity" but also on the flow of "acquired immunity → innate immunity" as a biological response to infection. For example, it has become clear that among memory T cells, some tissue-resident T cells remain in localised areas such as mucous membranes in preparation for re-exposure, and that cytokine production from these cells induces activation and migration of innate immune system cells, resulting in rapid local immune responses [12]. In the pathogenesis of DAD, there are two possibilities: the innate immune response may be activated from the onset, or it may become the main cause of inflammation after the acquired immune system becomes involved, that is, a small number of "acquired immune system" cells stimulate a large number of "innate immune system" cells and become the main cause of inflammation.
However, the mechanism by which bystander CD8 + T cells locate and attack target cells is still not fully understood. A possible mechanism is that of the natural killer group 2 member D (NKG2D)-NKG2D ligand (NKG2DL). NKG2D is expressed on the surface of CD8 + T cells and CD56 + natural killer cells, while NKG2DLs, such as MHC class I chain-related gene A/B (MICA/B), are expressed on the surface of injured cells. CD8-mediated cell injury via the NKG2D-NKG2DL mechanism has been reported in relation to a wide variety of pathological conditions, such as cancer immunity or infectious disease [13], but CD8-mediated endothelial cell injury via this mechanism remains to be fully elucidated. However, hypercytokinaemia induces the expression of NKG2D ligands in endothelial cells [14, 15], and this process may contribute to the development of DAD. If the mechanism by which bystander CD8 + T cells attack endothelial cells is elucidated, new therapies can be developed in the future.
In this study, it was found that CD8 + T cells predominantly infiltrated in the DAD lesions. In addition, many infiltrating CD8 + T cells seem to be GrB + /CD25-/PD-1-, a unique phenotype of antigen-independent activated CD8. In the organizing phase of DAD, the number of CD3 + and CD4 + cells was significantly increased compared to that in the acute phase of DAD, whereas the number of CD8 + and GrB + cells was not significantly different. In the organinzing phase of DAD, the number of CD25 + cells and PD-1 + cells was also significantly increased compared to that in the acute phase of DAD; this might be influenced by an increase in the CD4 + cells. The number of CD25 + or PD-1 + cells was considerably lower than that of CD3 + , CD4 + , CD8 + , or GrB + cells in the acute as well as organizing phase of DAD lesions. This suggests that CD8 + T-cells with the GrB + /CD25-/PD-1 phenotype may be present in the organizing phase of DAD in numbers not different from those in the acute phase of DAD. In conclusion, bystander CD8 + T cells may contribute to the development of DAD; however, whether they are the cause or effect of DAD remains unclear. The cellular immune response based on the innate immune system involving bystander CD8 + T cells may play an important role in the formation of lesions in DAD.
Acknowledgements
The authors would like to thank Professor Hiroyuki Kanno (Shinshu University) for his advice and encouragement, as well as Ms. Tomoko Nishizawa and Ms. Shizu Fujii for their technical assistance.
Author contributions
All the authors contributed to the study. Maki Ohya and Mikiko Kobayashi designed the study. Sample preparation and immunohistochemistry were performed by Maki Ohya, Ayako Tateishi, and Mikiko Kobayashi. Data were collected by Maki Ohya, Ayako Tateishi, and Mikiko Kobayashi. Maki Ohya analysed the data. Yuki Matsumoto and Hidetoshi Satomi assisted with the data analysis and interpretation. The first draft of the manuscript was written by Mikiko Kobayashi, and all the authors provided comments on the previous versions of the manuscript. All authors have read and approved the final manuscript.
Funding
This research was partially supported by a grant from JSPS KAKENHI [Grant Number JP 21K15399 to MK].
Data Availability
All data generated or analysed during this study are included in this published article (and its supplementary information files).
Declarations
Ethical approval
This study was approved by the ethics committee of Shinshu University School of Medicine, Japan (No. 5146) and the ethics committee of Marunouchi Hospital, Matsumoto, Japan (No.22–24).
Competing interests
None.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
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Data Availability Statement
All data generated or analysed during this study are included in this published article (and its supplementary information files).