Lymphocytes Studied by Raman Microspectroscopy

The Raman spectroscopy detects the interaction of the incident light with the electrons in the illuminated molecule. The use of Raman spectroscopy to investigate biological molecular structures and the recognition of their particular functional groups have been growing rapidly, and nowadays the use of Raman spectroscopy has expanded toward the cellular level. The activation of lymphocytes occurs when they are exposed to viruses or other foreign antigens. We have observed that Raman spectroscopy can be used to screen the activation of lymphocytes during viral infection. We have indicated the bands that reveal differences between activated and intact cells. The most important marker of the lymphocyte activation process is the prominent 521 cm − 1 disulfide band which marks the immunoglobulin formation. The blood from the patients with viral infections, e.g., mononucleosis, and from healthy volunteers was obtained by venipuncture during hospitalization in the University Hospital in Kraków.


Introduction
The proper functioning of the immune system results from close cooperation of the mechanisms of nonspecific (innate) immunity and adaptive immunity (acquired) [1]. Both mechanisms operate by direct contact between the cells and through interactions in which cytokines or chemical mediators participate.
The studies were conducted in accordance with the guidelines for good clinical practice (GCP) according to the ethical principles for medical research involving human subjects (Declaration of Helsinki). The study was approved by the local bioethical committee.

Single cell measurements by Raman spectroscopy
Raman spectra were collected using a Renishaw inVia spectrometer, working in confocal mode, connected to a Leica microscope. A 785-nm HP NIR (high-power near IR) diode laser and a 514.5-nm Ar + laser were used to excite the samples. The laser beam was focused by a 100-fold magnifying glass, the high-class Leica objective for standard applications, with a large numerical aperture (NA = 0.9). The laser power was kept low, ca. 1-3 mW at the sample, to ensure a minimum invasion of cells. Principal component analysis (PCA) was applied to determine the variance between the Raman spectra of a single lymphocyte of a patient with identified early stage of infectious mononucleosis and healthy donors. PCA was performed in the whole spectral region with the Unscrambler X software packages (v. 10.3, CAMO Software, Oslo, Norway). The Raman spectra were smoothed using a Savitzky-Golay smoothing algorithm (13 smoothing points), baseline corrected and unit vector normalized.
The first principal component, PC-1, contains the highest percentage of variation, which indicates the direction of the maximum variation in the dataset.

Raman spectra of naive lymphocytes
Lymphocytes carry out immune surveillance, that is, they constantly monitor tissues for the presence of foreign antigens [21]. The human body continuously produces new cells.
They circulate between blood and lymph, not only by lymphatic organs but also to a lesser extent by other body tissues [22]. It takes around 24 hours to complete the circulation. Circulating lymphocytes are a group composed of small, long-living cells. The continuous movement of these lymphoid cells means that they are always available for immune defense [3].
The circulatory route of naive lymphocytes T and B is different from that of the memory and effector lymphocytes. In the pool of circulating lymphocytes, the majority of cells are immune memory T cells [21]. The B lymphocytes account for only 15% of the total pool of circulating lymphocytes. Their circulatory pathways are still not well understood.
Cellular processes may be monitored using Raman spectroscopy exploiting the chemical specificity of using the high polarizability of certain functional groups that build molecular cell systems [22][23][24]. No staining is necessary to observe the diversity of areas inside the cell [25]. The  membrane of the cell (e.g., aq2) clearly differs from the center (e.g., aq13) ( Figure 1C and D); this variation is related to the lower presence of protein in the cell membrane, estimated by the intensity of the amide I band. The middle area is relatively homogeneous due to the distribution of proteins, which indicates fully mature cell ( Figure 1D). The significant Raman bands observed for human lymphocytes and their assignments are collected in Table 1.

Immunity against viruses
Viruses are usually small compared to other organisms that cause infection; their metabolism is closely dependent on the host's metabolism [26]. That is why viruses are not capable of replicating themselves. Therefore, the key process in viral infection is intracellular replication, which can even lead to the death of infected cells.
Antibodies prevent the penetration of the virus into an uninfected cell and thus limit some viral infections that spread with the blood.

Influenza and its etiology
Influenza is an acute illness of the respiratory system caused by influenza viruses that belong to the Orthomyxoviridae family. They are divided into three types A, B, and C on the basis of antigenic features of nucleoprotein and matrix protein antigens [27].
The host's response to flu infection is a complex system of dependencies between humoral immunity, local antibody production, cellular immunity, and other mechanisms. The presence

Infectious mononucleosis and its etiology
EBV virus induces an infectious mononucleosis characterized by heterophilic antibodies, fever, sore throat lymphadenopathy, and atypical lymphocytosis [28]. The virus belongs to the family Herpesviridae. The viral genome is a double-stranded DNA with a linear system, surrounded by a nucleocapsid with an icosahedral symmetry; on the outside there is a capsule containing glycoprotein.
More than 90% of adults report the presence of antibodies to EBV infection. An EBV is transmitted with saliva. The infection of B lymphocytes present in the tonsils crypts may take place directly; the next stage is blood virus spreading. There is observed lyphoid tissue growth due to EBV infected B lymphocytes proliferation and the accompanying T-cell reaction.

Raman spectrum of activated lymphocytes in response to the influenza virus
Once naive lymphocytes have been exposed, they are activated. This is an important element in the humoral immune response of the body. Free antibodies present in the blood, in the lymph, in all body fluids, and in secretions are involved in this process [1].
They are produced by B cells, initially functioning as APC, which, due to cooperation with Th2 lymphocytes, are transformed into clones of plasma cells producing antigen-specific antibodies [29]. Although antibodies do not destroy infectious agents but they bind epitopes in a specific way, so they are able to initiate effector mechanisms and eliminate the pathogen from the host organism.
That is why LeBien and Tedder commented that the discovery of B cells did not result from cell identification, but rather the identification of a protein, that is, immunoglobulin or antibody [30].
As a response to increased lymphocyte activation, a significant change in their shape and behavior is detected (compare Figure 1A and Figure 2A). Some of the stimulated B lymphocytes become active in the production of antibodies against foreign antigens, and they are transformed into plasma cells (compare Figure 1D and Figure 2D). This process allows the immune system to recognize infectious agents and prepare responses to them.
The spectroscopic marker of the lymphocyte activation process is a prominent 521 cm −1 disulfide band which marks the formation of the immunoglobulin (Figure 2C, aq41).
Immunoglobulin, initially present in the cytoplasm and then bound to the surface, is the main feature of B lymphocytes. They are used to identify specific antigens [26]. Immunoglobulins, regardless of function, have a similar molecular structure as well as basically identical mechanism of reacting with antigen [21]. They are all built from the same basic units, light (L) and heavy (H) polypeptide chains [31]. The basic immunoglobulin unit is a tetramer consisting of two identical heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds, in so-called hinge region, and each heavy chain is linked to a light chain by a disulfide bond (Figure 3). The disulfide bond is a structural feature of many significant biological molecules, because it provides additional stability to a protein molecule. Therefore, the structural characteristics of the CS▬SC dihedral angle and the structure of the entire molecule are important [32].
Raman spectroscopy is the best spectroscopic technique to monitor the disulfide bonds and the structure and conformation of a protein [33,34]. Compounds containing disulfide bonds usually show well-defined bands in the Raman spectra that arise from C▬S and S-S stretching modes that are also sensitive to the CS▬SC dihedral angle [32]. Additionally, they appear in a region of the Raman spectrum that is relatively free from other intense bands. The experimental Raman data can be correlated with X-ray data and the results obtained from calculations [35][36][37][38].
The spatial structure within the N-terminal section of the H and L chains makes it extremely important [21]. It is now known that one antibody molecule may bind to two or more completely different chemically determinants, which indicates that the anti-determinant does not directly recognize the specific chemical configuration of the antigen, but its overall spatial shape. Therefore, the role of Raman spectroscopy, which makes it possible to determine the structure, seems to be very promising.
Activation of lymphocytes, marked by the appearance of an immunoglobulin, also manifests itself as changes in the cell content. The peak of ca. 1130 cm −1 (protein C▬N str.) loses its intensity as well as 1100 cm −1 phosphate backbone vibration, indicating DNA concentration [9]. These spectral changes signify the lymphocyte evolution toward the plasma cell, which has a small eccentric nucleus, and most of the cell is filled by cytoplasm and well-developed rough endoplasmic reticulum (RER) where immunoglobulin synthesis takes place.
This is in agreement with the presence of intense lipid Raman bands at 1450 cm −1 in plasmocytes (because of RER).

Another important Raman band, amide I (C〓O stretching and N▬H bending vibrations)
at 1662 cm −1 , is more intensive ( Table 1). This position indicates that immunoglobulin in the intact form is predominantly composed of antiparallel β-sheet structure [33]. The protein composition seems to be different in activated form as amide I band intensity is slightly shifted toward lower wave numbers ( Table 1).
A further characteristic band, which occurs as well in naive lymphocytes as in the activated form, is the 850 cm −1 peak due to the exposed tyrosine residues in the proteins ( Table 1) [33].
Tyrosine is an important amino acid in signal transduction and regulating cellular activity (by tyrosine kinase) [39].

Lymphocytes
The principal component analysis (PCA) was applied to distinguish two areas of the B cell, classified as activated, and the one in which the presence of an immunoglobulin is not yet manifested (Figure 4). The PCA used to reduce the large amount of spectral information contained in the Raman spectra into a few principal component parameters. The activated area is characterized by PC-1 up to 61%.

Raman spectrum of activated lymphocytes in response to the EBV virus infection
The B cell activated in EBV infection and distribution of immunoglobulin is presented in Figure 5 (785 nm laser line) and  Table 1). Similar results were received for immunoglobulin in activated lymphocytes in influenza. The PCA model was built based on spectra recorded for lymphocyte B for the two research groups, at the beginning of hospitalization due to EBV infection and after receiving medical tests indicative of recovery ( Figure 7A) [40]. The PCA score plots show a separation based on differences in the lymphocytes' content for these two research groups.
Maxima bands in the loadings plot ( Figure 7B groups, counts only 1% variation for spectral dataset analyzed in the whole range. When a narrower range is analyzed, an important component becomes PC-1 that sums 37% of variation ( Figure 7C). Perhaps, this indicates the specificity of EBV pathogenesis and the formation of latent form in the B cell.

Conclusions
In the case of flu infection, the presence of antibodies in the serum can be confirmed by hemagglutination inhibition (HI) test, in complement fixation (CF) or neutralization reaction, and also using ELISA, the enzyme immunoassay [1]. Using these biochemical methods, antibodies are recorded only after 1 week. Raman spectroscopy gives this information, at the level of a single cell, immediately when single cell is activated.
Raman spectroscopy allows to identify a B lymphocyte. Once stimulated by binding to a foreign antigen, for example, virus, a lymphocyte is getting ready to multiply into a clone of identical cells.
On the other hand, it allows immediately to determine that B lymphocyte was in contact with the virus by the appearance of the 521 cm −1 marker band.
Antibody molecule may not directly recognize the specific chemical configuration of the antigen, but its overall spatial shape; therefore, the role of Raman spectroscopy, which makes possible to determine the structure, seems to be very promising.
The character of considered viral infections is different. In Raman spectra, we can observe it in PCA. The fact that only less than 40% for EBV is separated, between ill and control recovery groups, could be due to a latent form of EBV virus. In influenza the observations were different-there were about 60% PC factor-separating data.