The identification of immune cells began more than 150 years ago. The discovery of the dendritic cell in 1973 was a major breakthrough, and initiated a new era in cellular immunology. In recent years an increasing number of novel leukocyte subsets with specialized functions in the immune system have been described. These cells are categorized based on morphology and basic cellular functions like in vitro adherence, phagocytosis, cytotoxicity and suppressive functions. Today novel techniques like cell sorting, multicolor flow-cytometry, and RNA sequencing provides the immunologist with unique tools to dissect the cellular immune system.

Cellular immunology was born with the discovery of the microscope, the definition of the cell theory by Matthias Schleiden, Theodor Schwann and Rudolf Virchow (1821-1902), and the observation that pus cells are a mixture of dead tissue, bacteria, and white blood cells. Importantly, at that time the observation that an increased number of leukocytes in the blood is linked to pathology in the organism resulted in an increased interest in understanding the role of different leukocyte subsets.

Later, the improvement of the microscope by William Addison confirmed these observations by the demonstration of the diapedesis of white blood cells from the blood vessels into the tissue. Concepts like myeloid vs lymphoid cells soon followed based on the description of nuclear size and morphology, However, it was not until the emerging bacteriology around 1900 that a more detailed understanding of leukocyte subsets and their functional role in the immune system was achieved, in particular based on direct observations of phagocytosis performed by cells such as macrophages and granulocytes (1).

Although not standing alone, such relatively simple observations are still part of the discovery of novel cell subsets in immunology. In 1973, the simple in vitro discrimination of the ability of cells to adhere to glass and plastic was used for the groundbreaking definition of the dendritic cell (DC). During studies of mouse spleen cells and their adherence to glass and plastic, Steinman and Cohn identified the DC as a "large stellate cell with distinct properties from mononuclear phagocytes and granulocytes" (2). Based on the mixed leukocyte reaction (MLR) assay that involve co-culture of cells with allogeneic responder cells, Ralph M. Steinman demonstrated that DCs are the most potent cells for antigen presentation to T cells.

Figure 1. Ralph Marvin Steinman (1943-2011) discovered the immune system's sentinel dendritic cells, and demonstrated that science can fruitfully harness the power of these cells and other components of the immune system to curb infections and other communicable diseases. He received the Nobel Prize in Physiology or Medicine 2011 posthumously. Steinman was born in Montreal, Canada and received a B.S. degree from McGill University in 1963, and an M.D. from Harvard Medical School in 1968. He joined The Rockefeller University in 1970 as a postdoctoral fellow in the Laboratory of Cellular Physiology and Immunology, and was appointed professor in 1988. Steinman's research began as an attempt to understand the primary white cells of the immune system - the large "eating" macrophages and the exquisitely specific lymphocytes, which operate in a variety of ways to spot, apprehend, and destroy infectious microorganisms and tumor cells. His subsequent research pointed to dendritic cells as important and unique accessories in the onset of several immune responses, including clinically important situations such as graft rejection, resistance to tumors, autoimmune diseases and infections including AIDS. Photo: Rockefeller University. http://newswire.rockefeller.edu/2011/

The importance of these findings was recognized when the Nobel Prize in Physiology or Medicine 2011 was awarded, one half to Ralph M. Steinman "for his discovery of the dendritic cell and its role in adaptive immunity", and the other half jointly to Bruce A. Beutler and Jules A. Hoffmann "for their discoveries concerning the activation of innate immunity".

The DCs are key players in antigen recognition in the innate immune system and perform efficient antigen-uptake, processing, presentation on MHC molecules and priming, differentiation and imprinting of T cells. A large part of the important insight has been achieved in part through in vitro observations of DC and T cell co-cultures in MLR's.

At the time of DC discovery a link between activation of the innate immune system and the subsequent priming of cells of the adaptive immune system was still missing. MLR and other functional assays like phagocytosis assays, cellular cytotoxicity assays, migration assays, together with ELISPOT and limiting dilution assays for enumeration of antigen specific B and T cells allowed immunologists to study a spectrum of function in cellular immunology. They defined the capacity of novel leukocyte subsets in relation to functions including phagocytosis, NK and complement function and cytolytic activity, lymphocyte activation, cytokine secretion, chemokine dependent migration, and cytotoxic T cell (CTL) mediated cytolytic activity. However, an important modern strategy for immune cell discovery is the selection based on surface markers.

A classic example is the discovery of regulatory T cells with pivotal functions in immune homeostasis. They were identified based on surface expression of CD4+CD25 followed by sorting of cells and use in in-vitro co-culture with effector T cells to elucidate their suppressive function.

Improved methods for cell sorting combined with mass cytometry (CyTOF), have overcome the limitations of spectral overlap for conventional fluophore-based flowcytometry. With this advantage it is still likely that we will discover rare and specialized leukocyte subsets in the years to come, and obtain an improved understanding of their function by applying whole genome RNA sequencing. With such discoveries we may lever and fine tune the understanding of the immune system, exemplified by the current intense research in innate lymphoid cells subsets. However, the traditional protocols for cell extraction of solid tissues, including lymphoid organs, are still creating limitations forstudying immune cell diversity, since many cells are not extracted from lymphoid tissues, or they are stressed and destroyed by the extraction procedure. However, novel methods that combine immunohistochemistry with multiplexing of surface markers at the single cell level, have the potential of detailed cellular phenotyping, which could revolutionize our understanding of immune cells and their interactions in the immune system.

As for other immune cells present in low numbers in the blood, studies of DC immunobiology have been hindered by difficulties of obtaining sufficient numbers of cells. The in vitro protocols for the differentiation human peripheral blood monocytes cultured with GM-CSF and IL-4 into DCs were a major breakthrough in DC research in 1994 (3).

Figure 2. Dendritic cell. A dendritic cell passes information to T cell. Dendritic cells are the key antigen presenting cells of the immune system. They are the cells that initiate, direct and regulate the immune responses. Photo: http://www.dendriticcellresearch.com

These culture methods have allowed for improved understanding of DC immunobiology including the immature vs mature stage for tolerance induction vs immune activation, the link between innate antigen receptor ligation, DC cytokine secretion, and the priming of the increasing number of T cell subtypes such as TH1, TH2, TH9, TH17 etc. with different functions in the immune response. This field has recently been expanded with protocols that allow in vitro monocyte differentiation into various DC types as well as M1 and M2 macrophage subtypes.

Indeed, the in vitro differentiation of DCs combined with co-cultures of syngeneic or allogeneic T cells in MLR assays has advanced the ability to screen pharmacological compounds for inflammatory vs anti-inflammatory capacity, and for the ability to influence T cell priming in different directions, like TH1, TH2, TH17 etc. Using novel high throughput screening technology based on activity assays that apply differential scanning fluorimetry, fluorescence, absorbance, or microfluidic capillary electrophoresis, it is now possible to design suitable cell based assays (including DC assays) that can be screened against chemical library collections of more than 15,000 compounds at one time.

An important outcome of in vitro DC culture methods is the intelligent vaccine development strategy based on in vivo DC targeting. Thus, novel adjuvants can be tested and screened for effect on DC immunobiology, and the antigen can be tested for ability to target relevant receptors for uptake together with its route of antigen processing and presentation on MHC molecules to T cells. In example, we have characterized C-type lectin receptors on human DCs, and analyzed the binding of glycan-conjugated vaccine antigens, the intracellular location of such antigen and the capacity for DC cross-presentation of the vaccine epitope to specific CD4 and CD8 T cell hybridomas (4).

The protocols for human monocyte derived DCs also encouraged immunologists to develop DC based cancer vaccines based on the dogma that DCs are the best activators of tumor-specific CTLs, and that therapeutic DCs can bypass the tumor-mediated suppression of in vivo DCs in the tumor microenvironment (5). Researchers have invested massively in developing in vitro methods and validation for optimization of DC surface molecules, and cytokine profile of DCs to be used in cancer vaccines.

This insight has been translated to clinical trials where a large number of DC parameters have been tested, including vaccine administration route, DC maturity, antigen loading such as HLA class I binding peptides, tumor lysates and DC fused tumor cells. Most recently, this effort has resulted in a DC-based cancer immunotherapy against prostate cancer, Sipuleucel-T (Provenge) that is now approved by the FDA.

Based on the observation that immature in contrast to mature DCs may play a role in peripheral tolerance as opposed to induction of immunity, early translational clinical studies demonstrated that immature DCs pulsed with antigen resulted in removal of antigen specific IFN-gamma producing CD8+ T cells from the blood and replacement with immunosuppressive IL-10 secreting T cells. The possibility to in vitro manipulate DCs with the immunomodulatory cytokines TGF-beta and IL-10 to generate DCs with ability to induce T cell hypo-responsiveness to autoantigens was recently tested in a vaccine for patients with early onset diabetes. Thus, DCs are together with other immune cells being increasingly tested as cytotherapies including DC-based immunotherapy, adoptive T cell therapy, and NK cell therapy.

In conclusion, simple in vitro observations combined with modern sophisticated methods for cell sorting and phenotyping have the power to discover novel immune cell types. These advances may have implications for basic immunology as well as for improved cellular immunology assays for use in the pharmaceutical drug development, and even for cell-based immunotherapy on its own.