Tissues, not blood, are where immune cells function

Nature
Micrograph of a lymph node contains B cells (light blue), T cells (dark blue) and dendritic cells (yellow)

Immune cells have designated sites. In this lymph node, colours indicate cell types, such as B cells (light blue) and T cells (dark blue and green).Credit: Andrea Radtke

Early in the pandemic, my team spotted something surprising. When people were severely ill with COVID-19 and on a ventilator, the daily rinses of the plastic tubes in their windpipes contained immune cells from the airway. More surprisingly, what was in these airway samples was very different from what was found in the same patient’s blood.

The airway cells were producing high levels of cytokines — factors that recruit immune cells such as T cells to a tissue site and promote inflammation. By contrast, the corresponding blood samples were low in T cells, but high in other immune cells called monocytes, which were displaying unusual patterns of cell-surface receptors. Lung samples from patients who had died showed monocytes and a further type of immune cell (macrophages) clustered in the lung’s tiny air sacs; this is associated with the damage that typifies severe COVID-19. The unusual receptors suggested to us that monocytes circulating in the blood had been both altered and summoned by the cytokines produced in the airway1. Had we not collected both airway and blood samples, we would not have put these pieces together.

As this example shows, the pandemic has revealed major gaps in our understanding of the human immune system. One of the biggest is the reactions in tissues — at sites of infection and where disease manifests.

Immune cells are often referred to as white blood cells. But most, including more than 95% of T cells2, reside and function in tissues, particularly lymphoid organs — such as bone marrow, spleen and lymph nodes — and in barrier surfaces, such as the skin, gut and mucous membranes. Although infection with the SARS-CoV-2 coronavirus leads to virus-specific CD4+ and CD8+ T cells that are detectable in the blood for months or longer3, it is unclear what their presence in circulating blood means for tissue-based immunity in the lungs — or elsewhere.

Some immune cells are never found in blood. (Or rather, in many cases, we don’t know if they fail to enter the circulation or whether they change their properties when they do.) Some, such as macrophages, derive directly from fetal progenitor cells to mature in tissues such as the lungs, liver and spleen. Others, such as memory T cells, develop from activated T cells that migrate to tissues following priming in lymph nodes during an infection. These tissue-homing T cells take up long-term residence in tissues and can develop properties that are distinct in each.

To fully grasp the immune system, researchers need to understand respiratory, gut and skin immunity, and how each interacts with nearby lymph nodes. That means expanding support and infrastructure for obtaining tissues: forging alliances with clinicians, biobanks, hospitals and procurement agencies for donor organs. Here I describe how to make such research happen and what can be learnt.

Mouse models

Mouse models of infection, autoimmunity, cancer and inflammatory disease are extremely valuable for understanding the immune system. Mouse studies usually sample affected organs and associated lymph nodes, but rarely blood, owing to the tiny volumes involved. These site-specific studies have revealed fundamental processes — for instance, that respiratory infection with a virus prompts dendritic (antigen-presenting) cells to migrate from the lungs to adjacent lymph nodes, where they prime virus-specific T cells, such as CD4+ T helper cells. This priming promotes B cells to differentiate and produce antibodies; cytotoxic CD8+ T cells are also produced, and migrate to the lung to kill the infected cells and prevent viral spread. Once an infection is cleared, a small population of these virus-specific memory T and B cells persist in tissues, poised to launch protective responses rapidly if the pathogen is encountered again.

Micrograph of immune cells in tissue in the intestine's lymphoid tissue

Immune cells in the intestine are organized spatially: antibody-producing B cells (red) are surrounded by T cells (light blue).Credit: Takashi Senda

Mouse studies have also shown that, for site-specific viruses such as influenza or human papillomavirus, immunological memory is maintained by dedicated sets of memory T cells at the relevant site. These ‘tissue-resident’ memory T cells are found in the lungs for respiratory viruses, in the skin for cutaneous pathogens or in the female reproductive tract for genital infections4. Furthermore, these resident memory cells can protect against infection and can be generated by vaccines that target specific tissues, such as intranasal flu vaccines5.

In humans, it is not practical to follow an immune response from the start of an infection to the development of immunological memory. It’s not always clear where an infection starts or when, and sampling the relevant tissue over time is not straightforward.

However, humans have ample blood, which can be collected regularly. Blood contains all major lineages of immune cells, plus circulating antibodies and secreted cytokines. This approach has been invaluable for monitoring immune responses in real time, and for understanding the formation and function of antibodies and inflammation.

For SARS-CoV-2, examining blood has helped to track responses to infection and vaccines, and to find correlates of severe disease. But much of the story is still unknown, because the bulk of the immune action is in the tissues.

Lessons from tissues

Immune studies in tissues have led to therapies. In autoimmune diseases such as rheumatoid arthritis, a plethora of inflammatory markers become elevated in blood serum. When researchers looked specifically at the synovial fluid that fills the spaces between a person’s joints, they found high levels of the cytokine TNF-α, which was ultimately found to initiate this disease6. Now, anti-TNF-α blockers are among the most commonly prescribed medicines for rheumatoid arthritis and other inflammatory diseases, and are tremendously effective at treating symptoms.

Similarly, examination of immune cells in tumours that have been surgically excised or biopsied revealed functionally inactive T cells that failed to remove diseased cells7. This has led to the idea that many cancers can avoid immune defences. Such studies provided the rationale for the tumour immunotherapies that have revolutionized treatments for some breast, lung, colon and other cancers.

Micrograph of Spatial organization of immune cells within the lung

Spatial organization of immune cells in the lung. T cells (blue and orange) cluster around the major airway on the right, but not around the tiny air sacs where gases are exchanged in the blood.Credit: Stuart Weisberg

Our work and that of others indicates that the composition of immune cells is distinct in different tissues, with tissue-specific variation in gene expression, metabolic pathways and functional regulation8. Defining these properties could target therapies to tissue immune responses — but that requires first looking at the tissues.

Many tissue samples from living individuals can be collected during routine medical care. For instance, biopsies and elective surgeries allow collection of diseased and healthy tissue (see ‘Human tissues for immunology’). In surgeries from tonsillectomies to tumour removals, lymph nodes are removed and discarded. Cardiac surgeons typically remove and discard the thymus. Procedures to bypass the stomach and intestines remove gut tissues that are rich in immune cells and lymph nodes associated with the intestines. Such samples are particularly useful in showing the spatial organization of immune cells in these tissues, which can suggest how cell types communicate with and influence each other.

Human tissues for immunology

Source

How tissue obtained

Material available

Deceased individuals (multiple sites, one time point)

Autopsies (fixed tissues, no viable cells)

Many and variable: brain, liver, lungs, intestines, kidney, pancreas

Deceased individuals (multiple sites, one time point)

Organ donors (living cells that can be cultured and analysed for function)

Lungs, intestines, spleen, lymph nodes, bone marrow, pancreas, salivary glands, skin, thymus

Living individuals (single sites, single time point)

Surgery (tumour removal, gastric bypass, organ transplant, cosmetic surgery, gender reassignment)

Blood vessels, tumours, lungs, intestines, tonsils, skin, reproductive organs

Living individuals (single sites, single time point)

Clinical biopsies (for assessing cancer, infection, organ damage, monitoring a transplant)

Intestines, lungs, lymph nodes, pancreas, skin, breast, kidney

Living individuals (single sites, single time point)

Fine-needle aspirates (to assess cancer, infection)

Breast, liver, lymph node

Living individuals (single sites, multiple time points)

People treated for infections or respiratory distress; recipients of organ transplants

Respiratory washes from airway tubes, bronchoalveolar washes, biopsies (heart, liver, intestines)

Living individuals (single sites, multiple time points)

Healthy people, and people with infections, asthma, chronic lung diseases

Nasal washes, sputum, nasal swabs

Ten years ago, my team set up a new type of tissue resource to obtain samples from organ donors. We reached out to a local non-profit organization in New York City called LiveOnNY, which coordinates organ donation and collaborates with researchers and consenting families. As part of this collaboration, my team has an on-call surgeon ready to respond when the transplant coordinator alerts them that a potential donor has died.

After clinical teams obtain the person’s organs for transplantation, our surgeon collects tissues for research — including the intestines, lungs, many lymph nodes, the thymus, spleen, bone marrow, skin, tonsils and salivary glands — and brings everything directly back to the laboratory for processing and sample storage. Linking tissue collection with organ donation is the best way to preserve the most tissue and the most-viable samples. So far, results from these tissues match those of tissue from living patients9, although side-by-side comparisons are not always feasible.

We have so far obtained tissues from more than 500 donors. We have shared samples with many collaborating investigators and are profiling them as part of the Human Cell Atlas, which aims to create comprehensive reference maps of all human cells.

Research teams at the Wellcome Sanger Institute in Hinxton, UK, and the Karolinska Institute in Stockholm have set up similar programmes to obtain tissue samples. The University of Florida in Gainesville has established collaborations with many organ-procurement organizations, originally to obtain pancreas samples for type 1 diabetes research (the nPOD programme10).

Technological advances mean that RNA transcripts, protein content and gene modifications can be pinpointed even for single cells. Combined with computational analyses, these measurements allow the simultaneous identification of immune-cell composition, lineage and functional states. When applied to cells in blood, this detailed profiling has revealed immune signatures for certain infections and vaccines11, from flu to SARS-CoV-2.

My team and others have compared distinct tissues in hundreds of individual donors to map the different types of innate and adaptive immune cell in each8. Our ongoing studies include determining how tissue immune profiles correlate with factors such as sex and age.

Still, organ-donor samples provide snapshots of immune status at only a single point in time. One of the greatest challenges is to follow an immune response to infection, vaccination or other immune-based therapies in specific tissues. This would let us explore, for instance, the changes that occur with age or declining health status.

Micrograph of immune cells in the lymph node tissue

Immune cells in tissue are found at specific sites in the lymph node: antibody-producing B cells (green) are in circular follicles, partially surrounded by T cells (blue) with macrophages (red) on the periphery.Credit: Basak B. Ural

Three steps

What can researchers, institutions and funders do to advance work in human tissue immunity? I have three recommendations.

Promote paired sampling. Clinicians and immunologists should find more opportunities to pair the collection of blood and tissue, such as by obtaining respiratory washes from intubated patients or nasal washes from children and adults who have a respiratory disease. (Nasal washes are sufficiently non-invasive that they could also be used in matched healthy controls.) Paired samples could be taken from people undergoing diagnostic biopsies (such as during colonoscopy of the intestines) or research biopsies using fine-needle aspiration (such as from skin and lymph nodes).

These studies establish how immune parameters in blood and the relevant tissue correlate with each other, as well as with disease and disease outcome. For example, researchers studying a hepatitis B vaccine collected samples of lymph nodes near the injection site to compare the responses of immune cells there with what is in the blood12. A study of varicella zoster virus, which causes chickenpox and can reactivate to cause shingles, looked at T cells in the skin near the site of injection with a virus antigen13.

Approaches developed during the current pandemic mean that samples that it was not possible to analyse even a year ago can now supply in-depth data. This includes the nasal swabs used in SARS-CoV-2 testing, which contain enough cells for highly sensitive single-cell profiling. Gene expression can be measured in individual cells to help piece together the immune response. Those techniques can be applied to other sampling sites in other types of infection and disease, so that routine care can provide material for immune-based studies.

Adapt collections for biobanks. There are dozens of biobanks throughout the world; the largest contain samples from hundreds of thousands of people. Generally, however, these samples are not useful for immunologists. Instead of preserving samples chemically for pathology, or storing only DNA, plasma or serum, biobanks should also store frozen tissues and secretions full of immune cells and soluble factors. For example, studies of asthma and other chronic lung diseases have created biobanks of different respiratory secretions, including sputum, saliva, nasal swabs and bronchial brushings14. Studying the preserved immune cells in these samples could advance our understanding of mucosal immunity.

Support and streamline collaborations. Currently, an individual immunologist needs to connect with clinicians on a case-by-case basis to acquire tissue samples for research. Many institutions and centres have set up central cores for obtaining ethical or institutional-review-board approval, using clinical coordinators to get patients’ consent to take samples. However, these centres tend to be affiliated with clinical departments, not basic-science ones.

In the past, immunological research was conducted in pathology departments that received all clinical specimens, and distributed them to researchers. It is time to reinvigorate these old alliances and establish more and deeper connections between immunologists and clinicians who work with patients. Immunologists should pursue joint appointments in clinical departments. Clinician-scientists should train in immunology laboratories. Joint projects between the two fields should become common, and extend far beyond acquiring samples. There should be funding and programmes for immunologists to work with geriatricians to learn what leads to immune-system decline. Paediatricians and immunologists should be collaborating not just on childhood allergies, but also on understanding child immunity and the development of the immune system. Immunology should be pulled into many other disciplines not normally associated with it, such as neurology.

As SARS-CoV-2 continues to devastate the world, there is a new urgency to establish collaborations that can tackle intractable questions in human immunology. To move the field forward and translate findings to cures, immunologists must explore the whole body.

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