The 1980s were a critical and formative decade for the field of T cell biology. By the end of the 1970s, immunologists had discovered that there were different functional subsets of T cells (Th cells, cytotoxic T cells, and T suppressor cells), yet so much about these cells was unknown. For example, there was very little understanding about how T cells recognized Ag, how different subclasses of T cells could be identified reproducibly, and how T cells functioned at a molecular level. Of these unresolved issues, probably the most captivating mystery was the nature of the TCR, the cell surface receptor that T cells used to recognize specific combinations of Ag and MHC. By 1990, major strides had been made in resolving these questions, and T cell biology came into focus as a prominent and respected field.

After the genetic basis for Ab diversity was discovered in the late 1970s (1), identifying the nature of the TCR became a major priority for immunologists. There were many hypotheses about the type of receptor that T cells could use to simultaneously bind both diverse Ags plus self-MHC (2). Was the TCR a modified form of Ig, was it a unique protein related to Ig, or was it a completely novel protein complex? How could such receptors be generated in T cells? The first hint about the true nature of the TCR was the identification of a heterodimeric cell surface protein found on both mouse and human T cells in the early 1980s (3). However, the most fundamental questions about the generation of TCR diversity could not be settled until the genes encoding the TCR were identified. Cloning the genes for the TCR then became the “Holy Grail of Immunology,” and countless immunologists set out on a quest to seek this prize.

This quest led to an explosion of scientific discovery in the early 1980s that completely reshaped the field of immunology. In 1984, multiple articles describing the cloning of the genes encoding the α- and β-chains of the TCR were published (48). Researchers discovered that the TCR was essentially a T cell–specific relative of Ig, formed by two sets of rearranging gene elements each encoded by two different gene loci: α and β. The generation of the variable regions of the TCR β and α genes paralleled the rearrangement of V-D-J and V-J gene segments that recombine to generate the Ig heavy and L chain variable regions (911).

During the course of cloning the α and β TCR genes, a completely unexpected discovery was made: a third rearranging TCR gene without any known protein product or clear function (12). This new gene was called the TCR γ gene, and a new quest was born: to identify whether this new separate orphan TCR gene was expressed in a unique subset of T cells and whether this new receptor chain was partnered with the already identified TCR α- or β-chains or with a new, as yet unidentified TCR chain. As with the α-β TCR, the first hint of a new TCR was made at the protein level. Work with human PBLs and thymocytes identified a subset of CD3-expressing T cells that did not express the α-β TCR (13). Using Abs to CD3 and coimmunoprecipitation techniques, researchers discovered a novel protein heterodimer candidate for the new TCR, and it was tentatively termed the γ-δ TCR. Expression of this new γ-δ TCR correlated with an early stage of T cell development in the thymus, specifically a subset of CD3+CD4CD8 T cells whose development peaked in the fetal thymus, before the dominant emergence of TCR α-β–expressing thymocytes. However, as with the α-β TCR, true enlightenment about the nature of the TCR δ-chain would not come until the TCR δ gene was cloned.

This history sets the stage for the breakthrough Pillars of Immunology article by Chien et al. entitled, “A new T-cell receptor gene located within the alpha locus and expressed early in T-cell differentiation,” which was published in Nature in June 1987 (14). With this article, the α-β T cell versus γ-δ T cell paradigm was firmly established in the field of T cell biology, and a fascinating TCR α-β versus TCR γ-δ genetic program clicked into place.

The study described in the Chien et al. article started with a very clever bit of detective work. While looking at TCR α-chain rearrangements in thymocytes, the authors found unusual chromosomal rearrangements that repeatedly involved a region far upstream of the TCR α-chain C region gene. A technique used to map genomic organization before the era of deep sequencing, called “chromosomal walking” (using overlapping cosmid clones containing large fragments of genomic DNA), was performed to identify the new region of the TCR α-chain locus. Using a Southern blot probe specific for this new region, researchers observed a pattern of chromosomal rearrangements consistent with the expression of the putative TCR γ-δ protein heterodimer found in immature thymocytes and with TCR γ-chain gene rearrangements in early thymocyte development. In addition, an α-β TCR-expressing T cell hybridoma was found that had a rearrangement of this same region upstream of the TCR α genes. The rearranged genomic region was cloned out from the hybridoma and when analyzed was found to have a novel VDJ rearrangement. The appearance of the D region was very striking because the TCR α-chain did not use D region genes and consisted of only V to J gene rearrangements. This novel rearranged clone did not contain a C region; however, the VDJ exon is typically separated from the C region exons by ≥10 kb of DNA. Thus, the VDJ genomic probe was used to clone a cDNA from the T cell hybridoma that corresponded to a full-length mRNA of this novel TCR gene. When sequenced, a novel, fourth TCR C region was immediately apparent by its homology to other TCR constant regions genes and to Ig. This particular TCR gene had an out-of-frame rearrangement, which fit with the T cell hybridoma expression of an α-β TCR. This observation led to a search for a functional version of the new TCR gene. An in-frame rearrangement was soon cloned from a cDNA library made from CD4CD8 thymocytes, and the important question of whether this gene could encode a functional TCR protein was confirmed. This new C region was initially called “Cx,” but the cloned, full-length mRNA encoded a protein of roughly the same size as the δ-chain protein. Subsequent studies showed that Cx indeed encoded the previously identified δ-chain protein (15).

As revealed by Chien et al. and subsequent characterizations, the TCR δ gene locus was found to be situated right in the middle of what was originally thought to be the large α gene locus (11, 14, 16). It turned out that TCR α and δ genes both rearranged from a set of V gene segments located upstream of the unique D and J gene segments of TCR δ and far upstream of the unique J gene segments of TCR α (11, 14, 16, 17). This chromosomal arrangement predicted an elegant mechanism for allelic exclusion, such that an individual T cell could not express both an in-frame δ region gene and an in-frame α region gene, because rearranging the V and J gene segments to produce an α-chain V region would completely loop out and delete the TCR δ gene locus. This beautiful concept potentially plays a role in the early developmental switch between α-β T cells and γ-δ T cells. However, biology is never quite so simple (1820), and the complication is that α-β T cells can keep a TCR δ gene rearrangement on one chromosome and functionally rearrange the TCR α gene on the second chromosome. Indeed, this phenomenon was observed in the T cell hybridoma used to originally clone the δ TCR gene. Other mechanisms appear to play a role in the early α-β T cell and γ-δ T cell switch, such as TCR signal strength and Notch signaling during thymocyte development (2123). In addition, there is evidence for the programmed development of α-β T cells versus γ-δ T cells in the thymus (2426).

A notable feature of the large TCR α-δ gene locus is a strange quirk of evolution, wherein the TCR δ-chain gene that uses VDJ rearrangement is encoded in the middle of the TCR α gene, which uses only the simpler VJ rearrangement. This sort of idiosyncratic organization of rearranging gene elements is not seen with any other set of Ag receptor genes, either TCR or Ig, but may again relate to the need for α-δ allelic exclusion. In canonical TCR α-β and Ig light-H chain pairing, one chain is formed by V-J rearrangement and one chain is encoded by VDJ rearrangement. In a T cell where there is a functional TCR β gene rearrangement, the β-chain likely may pair more naturally with the VJ-encoded α-chain than a larger VDJ-encoded δ-chain.

This landmark discovery of the TCR δ-chain gene locus by Chien et al. not only resolved a major mystery in the T cell field but also brought γ-δ T cells forward as an exciting, new, and separate class of T cells that warranted further research. Despite their discovery 35 y ago, γ-δ T cells have remained the inscrutable oddballs of the T cell world. These peculiar T cells, with preprogrammed receptor specificities that can be activated by non-MHC ligands and products of metabolism (2729), are a fascinating and still unfolding part of T cell biology.

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The author has no financial conflicts of interest.