A fascinating new preprint shows that the immune cell status varies considerably over time, in healthy individuals going about their normal lives, even over a single day. This study contributes to building a basic database of immune responses that underline the unique nature of immunity in individuals.
Study: Intensive Single Cell Analysis Reveals Immune Cell Diversity among Healthy Individuals. Image Credit: Naumova Marina/ Shutterstock
The human immune system consists of immune cells designed to respond to a wide array of antigenic stimuli. The total of these responses makes up the individual’s immune profile. It is thus influenced by the medical history and lifestyle, the genetic background, and the place of residence. Most of what is known about these responses come from experiments on laboratory animals or sick people.
The occurrence of infection leads to various immune responses, mild or severe, at the site of infection. Circulating peripheral blood mononuclear cells (PBMCs) represent an important component of the immune profile, comprising innate immune cells such as monocytes (classical CD14+ and non-classical CD16+). Once they reach the infection site, they transform into macrophages or dendritic cells (DCs), engulfing the antigen.
These phagocytes travel to the local lymph nodes, where the antigen is presented on the cell surface to T lymphocytes, kicking off the adaptive phase of the immune response. Simultaneously, natural killer (NK) cells are activated to release cytotoxic proteins that eliminate the infected cells, enhancing the adaptive response.
Both B and T lymphocytes recognize a specific antigen by the bit called the epitope. The T cell receptor (TCR) contains VDJ segments comprising alpha and beta chains, each set of VDJ segments unique to the cell. T cell clonotypes have the same VDJ sequence.
Effector CD8+ T cells or cytotoxic CD8+ T cells are activated when they recognize antigens presented via class I major histocompatibility (MHC) molecules. These activate to kill the target cells.
B cell receptors (BCRs) are made up of immunoglobulin molecules on the outer surface of the cell. These can be any of a staggering array of receptors, rendered specific by the variability of the heavy and light chains of the immunoglobulins (IgK and IgL). Following antigen exposure, the naïve B cells differentiate into memory B cells or plasmablasts, the precursors of antibody-producing plasma cells.
Conventional methods such as flow cytometry, fluorescence-activated cell sorting (FACS), or bulk-RNA-seq have been used extensively to understand how PBMCs act as part of the underlying immune system. They can offer high-throughput gene detection over an extensive range of genes, but for the whole mass of cells rather than individual cells.
Secondly, they cannot help to reveal the VDJ sequences of each cell. In the current study, available as a preprint on the bioRxiv* server, the researchers opted to use single-cell RNA-seq (scRNA-seq) to observe single cells in detail as they encounter a specific antigen.
Such studies are already abundant, exploring single-cell responses to SARS-CoV-2 and their correlation with disease severity. However, in the current paper, the focus is on immunity among healthy individuals, including how the diversity of the immune response is acquired, sustained, and used.
In addition, the scientists describe the changes that occurred after vaccination with influenza and COVID-19 vaccines in the context of a healthy individual. The aim is to help delineate the fundamental diversity of immune responses among healthy controls typically used to study the immunological aspects of disease.
What did the study show?
Using highly reproducible and reliable data collection methods, the researchers found that while cell composition was in overall agreement with the expected patterns, there was a difference in individual profiles and at different time points within the same person. For instance, one had more B cells, while another had more NK and CD8+ T cells.
The exact profile suggested a bias towards humoral responses in the first but a T cell-dependent response in the second. Both subjects showed similar activation levels in NK and T cells and B cells, however.
In the first subject, represented as H1, plasmablasts were far more frequent than in other samples, while another B cell population was highest in H3 and H4. H7 had a unique profile with activated NK cells, perhaps because of the history of malignant B cell lymphoma in this older adult. NK cells are cytotoxic to tumor cells, and while inactive during the malignant phase, they recover to a reactive phase causing the disease to remit.
Even five years after chemotherapy, leading to the complete clearance of the malignant B cells, it is possible that the NK cell population that expanded during therapy remained high in number. Chronic stimulation or the presence of new somatic mutations can cause persistent expansion of NK cells, as sometimes seen after such patients recover.
Overall, the immunological diversity seems to be high in healthy people.
Over the course of a month, samples showed highly variable T cell clonotypes, with the top ten accounting for only a small percentage of the total cell population. These were unique to each subject, mostly derived from naïve T cells. This may indicate they come from a unique repertoire of naïve T cells waiting to be stimulated.
Over time, effector and memory CD8+ T cells dominated those clonotypes that were repeatedly identified, with a high frequency of mucosal-associated invariant T (MAIT) cells. A change from effector CD8+ T cells to memory CD8+ T cells was observed in one clonotype, perhaps because of a missed infection. It appears that T cell activation happens all the time, even when healthy.
The increases and decreases, in 85 and 209 immune cell clonotypes from two subjects, during this period confirmed the constantly shifting immune profile that results in a unique landscape over many years.
In one Indonesian subject, two clonotypes were found to be mostly for effector CD8+ T cells. When tested against cytomegalovirus and Epstein-Barr virus, both of which are common in such a setting, the scientists found high responses in the samples from this individual. The constant alerted state of the immune system in this subject may reflect the general trend in developing countries.
The BCR clonotypes also showed a non-overlapping profile, most prominently for the immunoglobulin heavy chain with 99% uniqueness. The first subject showed mostly IgM heavy chain unique clonotypes, indicating that this person might be encountering new antigens to which the B cells are constantly reacting. IgG heavy chains were more frequent among a small set of overlapping clonotypes, perhaps due to continued activation of this cell set.
Naïve BCRs are more diverse than memory B cell BCRs that have already been expanded by stimulation with a specific antigen. Knowing this, it appears that the increased diversity of BCRs in this subject is due to a higher naïve B cells frequency. This was found to be in the form of certain specific variants, each having clonotype diversity.
These findings showed that even though B cell responses dominate this individual, BCR patterns differ between individuals.
Exploiting the known antigenic profile and time of exposure of the flu shot and the COVID-19 vaccine, the researchers examined the immune cell profile after administering these vaccines to the same set of individuals. This showed differences in the response elicited in H1 and H2, with a common background.
Both showed an immediate classical monocyte expansion, with a subsequent decline in naïve B cell, γδ T cells, and CD4+ T cell frequency in peripheral blood, leaving other T cell populations intact. This was a temporary drop, however, recovering completely by the 28th day from vaccination. This is considered the time point when the immune response is complete, and memory cells have been established.
Individual differences such as a drop in MAIT cells in the initial phase and a greater decline in CD4+ T cells in one subject but not the other, coupled with an increased CD8+ T cells throughout. After vaccination, some sporadic and other persistent clonotypes were identified, the former being characteristically comprised of CD4+ and CD8+ naïve T cells.
Persistent T cells included both CD4+ and CD8+ memory T cells and MAIT cells, representing the early vaccine-induced clonotypes.
B cells were also found to respond to the vaccination. The features agreed with those reported by others, such as an early monocyte response with eventual complete recovery. These were more prominent with the COVID-19 vaccine than the influenza vaccine, especially the monocyte response to the former, perhaps because of its greater immunogenicity. Both non-classical and classical monocytes were induced in this case after the first and second doses.
The T cells responses in the persistent populations appeared to be in an activated phase, mostly induced after the first dose and then increasing in frequency after the second. These may be the cells that were specifically elicited by the COVID-19 vaccine.
Again, subject H7 showed fewer changes after vaccination, perhaps because of the subject’s age or because the immune activity centered on the NK cells was already high. Antibody responses were delayed but adequate, though lower than some others. Again, activated T cells were less affected, indicating that the baseline immune state determines the response to the vaccine.
What are the implications?
We revealed that the gene cellular components and gene expression profiles are diverse even in healthy individuals, possibly reflecting the personal history of previous immune responses.”
The influence of ethnicity on immune response can be traced through such studies to help uncover the inequalities in healthcare access and other aspects of health, as well as the social factors that underlie individual and community health status. By incorporating the healthcare records and environmental factors, the effect of ethnicity can be teased out.
Future studies investigating immune system fluctuations in disease should account for the baseline diversity amongst healthy individuals, as demonstrated in this study.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.