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Response of Peripheral Lymphocytes From Patients With Ossification of Posterior Longitudinal Ligament

Kanai, Yurika*; Kakiuchi, Terutaka**

Clinical Orthopaedics and Related Research: August 2001 - Volume 389 - Issue - p 79-88
SECTION II ORIGINAL ARTICLES: Spine
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The in vitro response of peripheral blood mononuclear cells or enriched CD4 + T cells from patients with ossification of the posterior longitudinal ligament to anti-CD3 monoclonal antibody has been studied. The response in both was significantly lower in patients with the continuous-type ossification than in patients with the segmental-type ossification and in healthy volunteers, and was inversely correlated with the number of vertebral bodies with ossified ligament. In patients with the segmental-type ossification, the response of peripheral blood mononuclear cells was significantly lower than that in healthy volunteers, but that of the enriched T cells was not. B cell proliferation in response to fixed Staphylococcus aureus cells was significantly lower in patients with the continuous-type ossification than in healthy volunteers but was not correlated with the number of vertebral bodies with ossified ligament. The B cell response in patients with the segmental-type ossification was not lower than that in healthy volunteers. Serum concentrations of transforming growth factor-β1 and basic fibroblast growth factor also were higher in patients with the continuous-type ossification than in patients with the segmental-type ossification and in healthy volunteers. The findings raise the possibility that continuous-type ossification of posterior longitudinal ligament might develop differently from segmental-type ossification.

From the *Department of Orthopedics, and the **Department of Immunology, Toho University School of Medicine, Tokyo, Japan.

Supported in part by Grants-in-Aid for scientific research to Terutaka Kakiuchi from the Ministry of Education, Science, Sports and Culture, Japan (No. 10670606).

Reprint requests to Terutaka Kakiuchi, MD, PhD, Department of Immunology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo, 143-8540, Japan.

Received: June 21, 1999.

Revised: July 31, 2000; December 18, 2000.

Accepted: January 4, 2002.

List of Abbreviations Used: bFGF basic fibroblast growth factor, BMP bone morphogenetic protein, HLA human lymphocyte antigen, HTLV-1 human T lymphotropic virus-1, IgA immunoglobulin A, IgG immunoglobulin G, IgM immunoglobulin M, IL-1 interleukin-1, IL-6 interleukin-6, TGF-β transforming growth factor-beta

Ossification of the posterior longitudinal ligament, which first was reported by Key, 8 causes myelopathy, radiculopathy, or both through compression of the spinal cord. Since Tsukimoto 23 described autopsy findings of ossification of the posterior longitudinal ligament, some investigators have conducted studies to clarify the mechanisms involved in ossification of the ligament. 16 The prevalence of the disease has been shown to be as high as 1.9% to 4.3% in Japan, 2.8% in Taiwan, and only 0.01% to 1.7% in the United States and Europe. 11 This difference has been interpreted to suggest that dietary habits could be a causative factor for ossification of the posterior longitudinal ligament. However, the prevalence of the disease in the United States and Europe recently has been reported to be as high as in Asian countries. 5

Based on the radiologic findings, ossification of the posterior longitudinal ligament was classified into four types: continuous, segmental, mixed, and localized. 24 The continuous-type is seen in 27.3% of the patients, the segmental-type in 39.0% of the patients, the mixed-type in 29.2% of the patients, and the localized-type in 7.5% of the patients. 24 The ossification is predominant in the cervical spine but is not confined to the cervical region. The ossification also was observed in the thoracic or lumbar region or both. The frequency of the association of the disease in the cervical spine with ossification in the thoracic or lumbar region or both is higher in the continuous-and mixed-types than in the segmental-and localized-types. 24

Possible causative factors proposed included diabetes, 20,24 hormonal imbalance, including estrogen 25 or parathyroid hormone, 19 and a generalized tendency toward calcification. 24 In addition, an allelic association of HLA markers DR2 and Bw62 with ossification of the posterior longitudinal ligament has been found, 17 and a genetic locus for the ossification was mapped close to the HLA region, 10 indicating involvement of genetic factors in the ossification. However, the mechanisms for the ossification remain to be clarified.

As local causative factors for ossification of the posterior longitudinal ligament, local production of several growth factors, including TGF-β1 and bFGF, has been suggested, 26,27 in addition to mechanical stress by abnormal mobility of intervertebral joints. 14 Recently, TGF-β1, bFGF, and BMP have been shown to induce bone formation, 1,3,18 whereas IL-1 and IL-6 have been shown to take part in bone absorption. 7,20 Transforming growth factor-β1 is known to inhibit lymphocyte response. 22 In this context, it is hypothesized that lymphocyte response might be affected during the development of ossification of the posterior longitudinal ligament through inhibition of lymphocyte function by these growth factors, interleukins, or both. Alternatively, lymphocyte response might be included in the development of ossification of the posterior longitudinal ligament through production of lymphokines affecting the production or function of the growth factors, interleukins, or both. In the current study, in vitro lymphocyte response was examined in patients with ossifications of the posterior longitudinal ligament. The findings suggest that either of the hypotheses is likely and raise the possibility that different types of ossification develop differently.

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MATERIALS AND METHODS

Patients and Clinical Findings

A diagnosis and classification of ossification of the posterior longitudinal ligament were made based on radiologic findings (Fig 1). 24 The patients were classified for analysis into two groups: segmental (including localized-type) and continuous (including mixed-type) types. 24 The number of the patients was 41 (27 men and 14 women; age range, 45–81 years, with a mean age of 61.6 years). Fifteen patients had a diagnosis of segmental-type disease, including two patients with localized-type ossification of the longitudinal ligament, and 26 patients had continuous-type ossification, including 16 patients with the mixed-type disease.

Fig 1A–D.

Fig 1A–D.

The control group consisted of 42 healthy volunteers, including 30 men and 12 women, with an age range of 41 to 77 years (mean age, 59.3 years). The severity of the disease was evaluated based on the Japanese Orthopaedic Association score of cervical spondylotic myelopathy (Japanese Orthopedic Association score; full score, 17). 13 The degree of ossification was evaluated based on the number of cervical vertebral bodies with an ossified ligament. All laboratory data, such as leukocyte count, ratio of lymphocytes or monocytes, and serum concentrations of IgG, IgA, IgM, C3, C4, and CH50, were within the normal range for patients with the disease.

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Preparation of Peripheral Blood Mononuclear Cells

From peripheral venous blood, mononuclear cells were separated by density gradient centrifugation on Separate-L (Muto Pure Chemicals Co, Ltd, Tokyo, Japan). The cells were washed and suspended in RPMI1640 culture medium (Sigma Chemical Co, St Louis, MO) supplemented with 10% fetal calf serum (Sanko Junyaku Co, Ltd, Tokyo, Japan) and 100 μg/mL kanamycin.

Where indicated, CD4 + T cells were enriched from peripheral blood mononuclear cells. Peripheral blood mononuclear cells were incubated on a plastic dish for 2 hours at 37° C to deplete adherent cells, followed by negative selection through a immunocolumn (Cellectplus human CD4 kit, Cytovax Biotechnologies Inc, Edmonton, Alberta, Canada) to deplete CD8 + T cells and B cells. The purity of CD4 + T cells was at least 93% based on cytometric analysis.

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Analysis of T Cell Response

For T cell response, a 96-well culture plate was coated with 0.07–5 μg/mL anti-CD3 monoclonal antibody (mAb) (X35, mouse IgG2a; Immunotech Co, Marseille, France), and then 2 × 10 5 peripheral blood mononuclear cells or 1 × 10 5 enriched CD4 + T cells in 250 μL culture medium were incubated on the anti–CD3-coated plate for 3 days at 37° C in 5% CO 2 in air. To assess proliferation of T cells, 0.2 μCi 3 H-thymidine (Amersham International plc, Little Chalfont, United Kingdom) was added to each well during the last 24 hours of culture, and the incorporation of 3 H-thymidine was determined on a Matrix 96 direct beta counter (Packard Instrument Co, Meriden, CT). The results were expressed as counts per 3 minutes. To analyze IL-2 production, 50 μL culture supernatant was harvested from each well and assessed for IL-2 activity using proliferation of IL-2–dependent CTLL-2 cells. Proliferation of CTLL-2 cells was assessed as described.

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Analysis of B Cell Response

B cell response was examined by incubating 2 × 10 5 peripheral blood mononuclear cells on a 96-well culture plate with 0.6 to 40 × 10 −4 % heat-killed and formalin-fixed Staphylococcus aureus cell suspension (Cowan I strain; Calbiochem-Novabiochem Co, San Diego, CA) at 37° C in 5% CO 2 in air for 3 days. Proliferation was assessed by the incorporation of 3 H-thymidine as described.

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Flow Cytometric Analysis of Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells were directly labeled with one of the following fluorescent isochiocyanate-conjugated mAb: anti-CD3 (T3; Coulter Co, Miami, FL), anti-CD4 (T4; Coulter Co), anti-CD8 (T8; Coulter Co), anti-CD19 (CLB-CD19; Nichirei, Tokyo, Japan), or anti-IL-2 receptor (2A3; Becton Dickinson Labware, Bedford, MA). The labeled cells were analyzed on a flow cytometer (Cytron Absolute Ortho Diagnostic Systems, Raritan, NJ).

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Measurement of Serum Concentration of TGF-β1 and bFGF

The concentration of TGF-β1 and bFGF in serum was determined using commercially available enzyme-linked immunosorbent assay kits (Amersham International plc), according to the manufacturer’s instructions. The detection limits were 16 pg/mL for TGF-β1 and 9 pg/mL for bFGF. For 28 of 39 healthy volunteers, two of 12 patients with segmental-type ossification of the posterior longitudinal ligament, and eight of 15 patients with continuous-type ossification of the posterior longitudinal ligament the concentration of bFGF was below the limit (9 pg/mL). In all of these cases, 9 pg/mL was used for the calculation of the mean value.

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Statistical Analysis

Results were expressed as mean ± standard deviation. For statistical analysis, an unpaired Student’s test and Pearson’s correlation coefficient were used. When the p value was less than 0.05, the difference or correlation was considered to be significant.

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RESULTS

Serum Concentrations of TGF-β1 and bFGF in Patients With Ossification of the Posterior Longitudinal Ligament

Serum levels of TGF-β1 in patients with ossification of the posterior longitudinal ligament were compared with levels in healthy volunteers. The concentration of TGF-β1 in patients with continuous-type ossification was 113.91 ± 72.85 pg/mL, which was significantly higher than the levels in patients with segmental-type ossification (65.75 ± 21.68 pg/mL) or the levels in healthy volunteers (62.31 ± 35.68 pg/mL) (p < 0.05). There was no significant difference between the concentrations in patients with segmental-type ossification and the concentrations in healthy volunteers.

Serum concentration of bFGF was 37.11 ± 59.35 pg/mL in patients with continuous-type ossification, a value that was significantly higher than the serum concentration in patients with segmental-type ossification (11.5 ± 3.0 pg/mL) and the serum concentration in healthy volunteers (10.03 ± 2.77 pg/mL) (p < 0.05). No significant differences were observed between the concentrations in patients with segmental-type ossification and the concentrations in healthy volunteers.

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In Vitro Response of Peripheral Blood Mononuclear Cells From Patients With Ossification of the Posterior Longitudinal Ligament to Anti-CD3 mAb

To compare in vitro T cell responses in patients with ossification of the posterior longitudinal ligament with responses in healthy volunteers, peripheral blood mononuclear cells were prepared from each individual and assayed for proliferation in response to anti-CD3 mAb. As shown in Figure 2A, proliferation of peripheral blood mononuclear cells was anti-CD3 antibody dose-dependent. The proliferative response in patients with the disease was lower than the proliferative response in healthy volunteers at any dose of anti-CD3 mAb tested. The response in patients with continuous-type ossification was significantly lower than the response in healthy control subjects (p < 0.01) or the response in patients with segmental-type ossification (p < 0.05). The response of peripheral blood mononuclear cells from patients with segmental-type ossification also was lower than the response of such cells from healthy volunteers (p < 0.05). The above p values were obtained at 0.3 μg/mL anti-CD3 mAb or higher.

Fig 2A–B.

Fig 2A–B.

Interleukin-2 production in the responses also was examined. As shown in Figure 2B, IL-2 production was detected at 0.6 μg/mL anti-CD3 mAb and reached a plateau at 1.2 μg/mL, although a proliferative response was detected even at 0.075 or 0.15 μg/mL (Fig 2A). Interleukin-2 production in the response of peripheral blood mononuclear cells from patients with segmental-or continuous-type ossification was much lower than that from the cells of healthy volunteers (p < 0.01 at 0.6 μg/mL anti-CD3 mAb or higher). No significant differences were observed between patients with segmental-type ossification and patients with continuous-type ossification.

To examine the possibility that the lower response to anti-CD3 mAb was attributable to the lower number of CD4 + T cells, peripheral blood mononuclear cells were analyzed in terms of the ratio in each subpopulation. Ratios of CD3 + T cells, CD4 + T cells, and CD8 + T cells in patients with ossification of the posterior longitudinal ligament were approximately equivalent to the ratios in healthy volunteers (65.6%, 41.3% and 25.9%, respectively, for the patients; 70.5%, 40.0% and 31.5%, respectively, for healthy volunteers). The ratio of CD25 + cells in patients with the disease also was very close to the ratio in healthy volunteers (7.4% and 8.9%, respectively). The ratios did not significantly differ between peripheral blood mononuclear cells from patients with continuous-type ossification and ratios from patients with segmental-type ossification (data not shown). Thus, the lower responsiveness to anti-CD3 mAb was not attributable to the lower ratio of CD4 + T cells.

The decrease in proliferation of peripheral blood mononuclear cells in response to anti-CD3 mAb was not found to be correlated with the severity of the symptoms evaluated by the Japanese Orthopedic Association score 13 (data not shown), but rather was inversely correlated with the number of cervical vertebral bodies with ossified ligament (Fig 3A). The inverse correlation was observed in patients with continuous-type ossification but not in patients with segmental-type ossification (Fig 3B, C).

Fig 3A–C.

Fig 3A–C.

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Response of CD4 + T Cells Enriched From Peripheral Blood Mononuclear Cells to Anti-CD3 mAb

CD4 + T cells were enriched from peripheral blood mononuclear cells and stimulated with anti-CD3 mAb. As shown in Figure 4, the proliferative response of CD4 + T cells from healthy volunteers was detected at 1.25 μg/mL anti-CD3 mAb and reached a plateau at 5 μg/mL. The proliferative response of CD4 + T cells from patients with continuous-type ossification was detected at 2.5 μg/mL anti-CD3 mAb and reached a plateau at 5 μg/mL, a level that clearly was lower than the level observed for healthy volunteers. In contrast, CD4 + T cells obtained from patients with segmental-type ossification responded as well as the cells from healthy volunteers and reached a plateau at 5 μg/mL anti-CD3 mAb. However, the responses at 1.25 μg/mL and 2.5 μg/mL were lower than the responses observed for healthy volunteers. Proliferation of CD4 + T cells from patients with ossification of the posterior longitudinal ligament was inversely correlated with the number of cervical vertebral bodies with ossified ligament (Fig 5A) but not with severity, as expressed by the Japanese Orthopedic Association score 13 (data not shown). However, no significant correlation was observed between proliferation and the number in patients with continuous-type ossification or patients with segmental-type ossification (Fig 5B, C). The number of patients examined might have been too small to obtain significant correlation.

Fig 4.

Fig 4.

Fig 5A–C.

Fig 5A–C.

In determining the response of CD4 + T cells, IL-2 production response also was examined. The results varied from experiment to experiment in terms of the dose of anti-CD3 mAb required for IL-2 production or the CTLL-2 cell proliferation counts, even in CD4 + T cells from healthy volunteers. However, in every experiment comparing IL-2 production in patients with ossification of the posterior longitudinal ligament with that in healthy volunteers, IL-2 activity in the culture supernatant of anti–CD3-stimulated CD4 + T cells from patients with the disease always was lower than that from healthy volunteers (data not shown).

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Proliferative Response of B Cells in Peripheral Blood Mononuclear Cells Against Staphylococcus aureus Cowan I

In vitro proliferation of B cells in peripheral blood mononuclear cells also was examined in response to Staphylococcus aureus Cowan I fixed cells. As shown in Figure 6, the proliferative response of B cells in patients with continuous-type ossification was significantly lower than that in healthy volunteers. However, the response of B cells in patients with segmental-type ossification did not differ significantly from that in healthy volunteers or in patients with continuous-type ossification, although the response seemed to be higher than that in patients with continuous-type ossification (Fig 6).

Fig 6.

Fig 6.

The observed decrease in proliferative response of B cells in peripheral blood mononuclear cells from patients with ossification of the posterior longitudinal ligament did not reflect the severity of the symptoms or the degree of ossification, given that no significant correlation was observed between proliferation at 4 × 10 −3 % Staphylococcus aureus Cowan I fixed cells and the Japanese Orthopedic Association score 13 or the number of cervical vertebral bodies with an ossified ligament. This trend was observed even in patients with continuous-type ossification (data not shown).

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DISCUSSION

In the current study, the in vitro response of lymphocytes was analyzed from patients with ossification of the posterior longitudinal ligament. T cell proliferation in response to anti-CD3 mAb was significantly lower in peripheral blood mononuclear cells or in enriched CD4 + T cells from patients with continuous-type ossification than that from patients with segmental-type ossification and healthy volunteers. In patients with segmental-type ossification, the response of peripheral blood mononuclear cells to anti-CD3 mAb also was lower than that in healthy volunteers, but the proliferative response to enriched CD4 + T cells was as high as that in healthy volunteers. In the patients with continuous-type ossification, the T cell proliferative response was inversely correlated with the number of vertebral bodies with an ossified ligament, but not in patients with segmental-type disease. The findings raised the possibility that continuous-type ossification of the posterior longitudinal ligament might be a distinct disease or etiologically different from segmental-type ossification of the posterior longitudinal ligament. As the current authors discuss here, most of the findings are consistent with the possibility.

The ratios of CD4 + T cells in peripheral blood mononuclear cells was approximately equivalent in patients with continuous-type ossification of the posterior longitudinal ligament, in patients with segmental-type disease, and in healthy volunteers, a fact that suggests the lower response to anti-CD3 mAb in peripheral blood mononuclear cells from patients with ossification of the posterior longitudinal ligament was not attributable to the smaller number of CD4 + T cells. In patients with continuous-type disease, the lower response of T cells in peripheral blood mononuclear cells might be because of the reduced ability of T cells, given that the response of enriched CD4 + T cells also was lower in these patients than in patients with segmental-type disease or in healthy volunteers. In contrast, the lower proliferation of T cells in peripheral blood mononuclear cells from patients with segmental-type disease than that in healthy volunteers seems not to be because of the reduced ability of the T cells, given that the response of enriched CD4 + T cells in patients with segmental-type disease was not lower than that in healthy volunteers. In these patients, other cells in peripheral blood mononuclear cells might regulate proliferation of CD4 + T cells in response to anti-CD3 mAb. Thus, T cells might be affected in patients with the continuous-type disease but not in patients with the segmental-type disease.

In IL-2 production, response to anti-CD3 mAb in peripheral blood mononuclear cells from patients with continuous-type ossification of the posterior longitudinal ligament and that from patients with segmental-type disease were lower than that from healthy volunteers. The reason the response of peripheral blood mononuclear cells from patients with segmental-type disease to anti-CD3 mAb was higher in proliferation than that from patients with continuous-type disease, and yet similar in IL-2 production, remains unknown. One possible explanation is that T cells from patients with segmental type-disease responded to anti-CD3 mAb and produced IL-2, which was consumed by the responding T cells.

The number of vertebral bodies with an ossified ligament was four or less in patients with segmental-type ossification of the posterior longitudinal ligament. In patients with the continuous-type disease, the number ranged between two and seven, and was greater than four in more than ½ the patients. In patients with the segmental-type disease, the distribution in each case was very wide compared with that in patients with the continuous-type disease (Fig 3). These distribution patterns might imply a difference in the two types of ossification of the posterior longitudinal ligament.

Transforming growth factor-β1 might play a role in decreasing the ability of T cells to respond to anti-CD3 mAb. An increased production of TGF-β1 has been found histologically in ossification of the posterior longitudinal ligament lesions. 27 Transforming growth factor-β1 is known to inhibit lymphocyte response, including T cell response. 21 However, in vitro T cell proliferation was not inhibited by TGF-β1 at the concentration observed in patients’ sera (data not shown). It is conceivable that an increased production of TGF-β1 at the lesions of the ossification affects the immunocompetency of peripheral lymphocytes as they recirculate near the lesions, a region where the TGF-β1 concentration is likely much higher than it is in serum. As suggested by the finding that an increased level of serum TGF-β1 was observed in the current study in patients with continuous-type ossification of the posterior longitudinal ligament but not in patients with the segmental-type disease, TGF-β1 production at the lesions of the ossification might be higher in patients with continuous-type disease than in patients with segmental-type disease. Supporting the role of TGF-β1 in decreasing in vivo T cell response, increased production of TGF-β1 has been shown to associate with decreased function of T cells in chronic fatigue syndrome. 2,4,9

In addition to inhibiting lymphocyte function, TGF-β1 has been shown to stimulate fibroblast proliferation and to increase matrix protein synthesis. 15 These TGF-β1 functions may be causative factors for ossification of the posterior longitudinal ligament. Thus, increased TGF-β1 production may participate in the development of ossification of the posterior longitudinal ligament and affect lymphocyte function, especially in patients with the continuous-type disease. Consistent with this possibility, higher frequency of the association with ossification of the posterior longitudinal ligament at the thoracic or lumbar regions or both has been shown in patients with continuous-type ossification than in patients with segmental-type ossification. 24

The observed affected responsiveness of T cells might have been one of the causative or accelerating factors for ossification of the posterior longitudinal ligament. Human T lymphotropic virus-1 infection has been shown to be significantly associated with ossification of the posterior longitudinal ligament, a finding based on an epidemiologic analysis in HTLV-1 endemic areas in Japan. 12 Human T lymphotropic virus-1 infects CD4 + T cells, thereby affecting their function. 6 However, the mechanisms for the development of HTLV-1 infection with decreased responsiveness of CD4 + cells into the ossification remain unknown. Thus, affected CD4 + T cell function by unknown causes may represent at least one of the risk factors for development of continuous-or mixed-type ossification of the posterior longitudinal ligament.

The pathologic ossification of the posterior longitudinal ligament has been shown to have a genetic background, and a predisposing locus for the ossification has been identified on chromosome 6p, close to the HLA complex. 10 The development of the ossification also has been shown to be associated with HLA. 17 However, in the current study, HLA typing was not done. The response of the T cells used in the current study was against anti-CD3 mAb stimulation. Thus, HLA molecules were not included directly in this type of T cell response. The locus might regulate T cell function directly or indirectly through unknown mechanisms.

Decreased responsiveness of peripheral T and B cells in patients with ossification of the posterior longitudinal ligament has been described. Additional research into immune function in patients with ossification of the posterior longitudinal ligament might provide new insights into the development of preventative measures and treatment of ossification of the posterior longitudinal ligament.

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Acknowledgments

The authors thank Toru Suguro, MD, PhD, and Yukikazu Okazima, MD, PhD, for encouragement, and Yayoi Okada, PhD, for technical assistance.

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Glossary

  • Anti-CD3 mAb and CD4 + T cells = Monoclonal antibody raised against CD3 molecules on the surface of T cells is used to detect T cells or stimulate them to proliferate or to produce IL-2. The majority of cells responding to the antibody are so-called helper T cells bearing CD4 molecules on the surface, which are called CD4 + T cells.
  • CD8 = T cells in peripheral blood usually bear CD4 or CD8 molecules on the surface. T cells bearing CD8 molecules are called CD8 + T cells and are so-called killer T cells.
  • CD19 = This molecule is one of those primarily expressed on the surface of B cells.
  • CD25 = This molecule is IL-2 receptor α chain, which is expressed on the surface of activated T cells.
  • Staphylococcus aureus Cowan I and B cells = This bacterial strain has been fixed and widely used to stimulate human B cells to proliferate. B cell is a major subpopulation of lymphocytes that differentiates into an antibody-producing cell under the appropriate conditions.
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