BS3A.78.pdf
Biomedical Optics 2014 © OSA 2014
Study of 3D Structural Differences between CD4+ and CD8+ T lymphocytes W. Jiang1, H. Hong2, R. Juskevicius2, D.A. Weidner3, Y. Feng4,1, L.V. Yang5, J.Q. Lu1, X.H. Hu1* 2
1 Department of Physics, East Carolina University, Greenville, NC 27858a Department of Pathology and Laboratory Science, Brody School of Medicine, East Carolina University, Greenville, NC 27834 3 Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834 4 Department of Radiation Oncology, Brody School of Medicine, East Carolina University, Greenville, NC 27834 5 Department of Oncology, Brody School of Medicine, East Carolina University, Greenville, NC 27834 *email:
[email protected]
Abstract: We acquired confocal image stacks from human CD4+ and CD8+ T lymphocytes extracted from spleen tissues to reconstruct and quantify their 3D morphology. Statistically significant differences in nuclear volume were found between the two subtypes. OCIS codes: (170.1530) Cell analysis; (180.1790) Confocal microscopy
1. Introduction T lymphocytes are an important component of mammalian immune response to pathogens and abnormal cells. T lymphocyte can be further divided into subtypes through immunophenotyping with fluorochrome-conjugated CD surface markers which have been shown to play different and critical roles in immune response. For example, CD4+ cells can be activated to become T regulatory (TReg) cells while CD8+ cells can become T cytotoxic (TC) cells. The TC cells kills infected and cancer cells carrying antigens or mutations. In contrast, the TReg cells perform regulatory roles by suppressing immune responses to self-antigens or deemed as “excessive”. Clinical studies of various T cell subtypes in cancer patients have shown that cancer patients tend to have higher ratios of CD8+/CD4+ cells [1, 2]. Despite the ability to classify T lymphocytes into subtypes using CD markers, development of label-free and rapid methods to distinguish these cells would yield powerful tools for study of lymphocytes and other white blood cells in immunology and other fields such as immunotherapy of cancer patients [3]. Besides its clinical implications, investigation of cellular structures among the different T lymphocyte subtypes provides insights on the fundamental relations between structure and function at the cell level. Over the past years, we have developed a diffraction imaging flow cytometry (DIFC) method which allows classification of biological cells based on the feature parameters extracted from the diffraction image data [4, 5]. It has been long known that strong correlations exist between the feature parameters of the diffraction images recording the spatial distribution of the coherently scattered light and the intracellular distribution of the refractive index [6-9]. Consequently the DIFC method makes it possible to distinguish cells with subtle differences in cells’ 3D morphology without the need to stain the cells [10, 11]. To lay a ground work for future study of T lymphocyte classification with the DIFC method, we have measured T lymphocytes’ 3D morphology through confocal imaging and reconstruction. Here we present the results of measurement and analysis of the morphological differences among the two subtypes of CD4+ and CD8+ T lymphocytes. 2. Methods Fresh human spleen tissues were obtained from the Department of Pathology, Brody school of Medicine at East Carolina University under an IRB approved protocol for study of human tissues. The tissue samples were kept on ice and transported to a cell laboratory in the same building for extraction of cells. A spleen tissue was first cut into small pieces in RPMI 1640 medium and a single suspension of splenocytes were generated by grinding the tissue piece with two frosted glass slides in the medium. The cell suspension was then centrifuged at 1500 RPM for 5 minutes into a pellet. After removing the supernatant and re-suspension, the red blood cells were removed by adding the red blood cell lysis buffer and shaking at room temperature for 10 minutes. The final suspension sample of 3 ml usually contains about 15 million splenocytes in PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA). The suspension sample of the extracted splenocytes was divided into aliquots for staining with different CD markers and subsequent sorting. Antibody markers of CD4 (Life Technologies, MHCD0418) and CD8 (Life Technologies, MHCD0801) were used to select CD4+ and CD8+ T lymphocytes from the prepared splenocyte suspension using a cell sorter (FACSVantage SE, BD). Each antibody marker was added to an aliquot followed by mixing and incubation on ice for 30 minutes. The prepared cell sample was washed with 2 ml of PBS/BSA and centrifuged at 1500 RPM for 5 minutes. After removing the supernatant, the stained cell pellet was re-suspended in 0.3 ml of PBS/BSA before
BS3A.78.pdf
Biomedical Optics 2014 © OSA 2014
sorting. A double sorting procedure was employed to ensure the purity of the obtained CD4+ and CD8+ T lymphocytes, whose number ranged from 0.5 to 0.7 millions in the final samples. The prepared T lymphocytes were double stained for fluorescent imaging with the Syto 61 dye for nuclei and MitoTracker Orange dye (both from Life Technologies) for mitochondria. After incubation in media with the two dyes for 30 minutes, the T lymphocytes were washed and brought to a confocal microscope (LSM510, Zeiss) on a glass slide for confocal imaging with an 100x oil-immersion objective. The fluorescence of Syto 61 dye was recorded into the red channel while the MitoTracker Orange fluorescence into the green channel of the output image files. For each sorted T lymphocyte suspension we randomly selected single cells to acquire one image stack of about 60 to 80 image slices, separated by 0.5μm, per imaged cell and build their 3D structures. A Matlab (7.1, Mathworks) based software has been previously developed [12] and applied to reconstruct and analyze the 3D morphology of the imaged cells from the confocal image stack data. The software algorithm consists of three steps: segmentation of intracellular organelles of cytoplasm, nucleus and mitochondria in each image slice of the green and red channels, slice interpolation and 3D reconstruction. After the reconstruction, 3D morphology of the imaged cells were analyzed by performing voxel based calculation of volumes, surface areas, shape and their ratio for each organelle of the imaged cells. Detailed descriptions of the image reconstruction and analysis are given in [12]. 3. Results We have imaged a total of 59 CD4+ and 44 CD8+ T lymphocytes from the extracted splenocytes to quantitatively characterize and compare their 3D morphology. Fig. 1 presents two sets of CD4+ and CD8+ T lymphocytes with a confocal image slice and a perspective view of the reconstructed 3D structure for each of the three cells in each set.
A
B
.
V=76µm3 Vrnc=77.27% Vrmc=0.61%
V=84µm3 Vrnc=84.29% Vrmc=0.48%
V=87µm3 Vrnc=85.17% Vrmc=0.45%
V=95µm3 Vrnc=87.3% Vrmc=0.16%
V=95µm3 Vrnc=79.53% Vrmc=0.15%
V=97µm3 Vrnc=83.68% Vrmc=0.14%
Fig. 1 Three CD4+ (A) and CD8+ (B) T lymphocytes with one confocal image slice in the top panel and a perspective view of the reconstructed 3D morphology in the middle. The three parameters at the bottoms for each cell are cell volume V, volume ratio of nucleus-to-cell Vrnc and volume ratio of mitochondrion-to-cell.
After 3D reconstruction, morphology of the three organelles of cytoplasm, nucleus, and mitochondria in each cell were quantified by their volume V, surface area S, perimeter and their ratios. Furthermore, the shape of the cell, nucleus, mitochondria and their centroid locations can be analyzed through various parameters such as sphericity, index of surface irregularity and the distribution of the distances of surface voxels from the centroid [12]. A total of 29 morphological parameters have been obtained and a two-sample t-test was performed with the SPSS software (Version 19, IBM) to investigate the structural differences between the two T lymphocyte subtypes. Values of selected morphological parameters are listed in Table 1. Careful analysis of the data in Table 1 show that while the cell morphology of the two T lymphocyte subtypes exhibit high similarity, most of the statistically significant differences (the lines with p < 0.05 in Table 1 are marked with red color) are related to the nucleus. Since the ratio of volumes between the nucleus and cell is high (close to 80%), this can in turn lead to some differences in the cell as shown by the shape of the cell. These results provide some useful insight into the structure-function relation of the T lymphocytes. For example, it may suggest that the CD8+ T cells, which can become cytotoxic upon activation, may require statistically significant larger nuclear volumes to express molecules needed for performing their function and exerting the higher level of activity in the context of immune response.
BS3A.78.pdf
Biomedical Optics 2014 © OSA 2014
Table 1 Morphological parameters of CD8+ and CD4+ T lymphocytes (line with p < 0.05 are marked in red) mean ± standard deviation Parameter Symbol Unit CD8+ T (n=44)(1) CD4+ T (n=59)(1) Cell volume Vc (2) μm3 92.90±18.0 88.82±19.5 Cell surface area Sc (3) μm2 14.92±4.64 13.02±4.37 Surface to volume ratio of cell SVrc μm-1 0.1639±0.05 0.1566±0.07 Index of surface irregularity of cell ISIc (4) μm-1/2 306.6±41.6 252.5±42.4 Average distance of cell membrane voxels to μm 3.274±1.45 3.558±1.30 centroid Standard deviation of Rc ΔRc μm 1.208±0.410 0.9290±0.217 Nuclear volume Vn μm3 72.56±12.1 65.02±14.0 Nuclear surface area Sn μm2 13.77±4.87 11.66±4.06 Nuclear perimeter μm 1929±457 1720±626 Index of surface irregularity of nucleus ISIn μm-1/2 267.3±89.9 294.7±77.1 Surface to volume ratio of nucleus SVrn μm-1 0.2278±0.057 0.2822±0.113 Sphericity of nucleus SPn 0.9770±0.152 1.102±0.255 Distance between the centroids of nucleus and cell Dnc μm 0.2235±0.118 0.2592±0.135 Volume ratio of nucleus to cell Vrnc 0.7901±0.097 0.7475±0.142 Average distance of nuclear membrane voxels to μm 4.825±3.38 4.752±3.08 centroid Standard deviation of Rn ΔRn μm 1.014±0.479 0.9957±0.513 Mitochondrial volume Vm μm3 1.11±0.489 0.91±1.12 Mitochondrial surface area Sm μm2 1.595±3.61 1.605±6.59 Surface to volume ratio of mitochondria SVrm μm-1 2.123±3.17 2.139±9.41 Index of surface irregularity of mitochondria ISIm μm-1/2 1332±1059 494.4±1020 Volume ratio of mitochondrion to cell Vrmc 0.0111±0.008 0.7657±0.126 (1) n = number of imaged cells, p is based on a two-sample t-test method. (2) V = Nv⋅V0 with Nv as the number of voxels inside the organelle of interest and V0 as voxel volume. (3) S = Ns⋅S0 with Ns as the number of voxels on the membrane of the organelle and S0 as the side surface of voxel. (4) ISI = Ns⋅a0/(V)1/2 with a0 as the side length (=0.07μm) of voxel.
p(1) 0.282 0.036 0.543 3.85x10-9 0.085 2.20x10-5 0.005 0.019 0.063 0.099 0.004 0.005 0.164 0.090 0.909 0.855 0.270 0.993 0.991 9.88x10-5 0.890
4. Conclusion We have applied a previously developed imaging method to measure and analyze the 3D morphology of two important T lymphocyte subtypes. Their morphology has been quantitatively characterized and compared. The results not only provide insight into the structure-function relation of T lymphocytes but also prepare the ground for future development of label–free methods to classify these cells using the DIFC method [10, 11]. 5. References [1]
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