Nanos gigantum humeris insidentes. (Like dwarfs sitting on the shoulders of giants.) —Bernard de Chartres (1120) If I have seen further than others, it is by standing on the shoulders of giants. —Isaac Newton (1676)

The immune system is the guardian of our organismal integrity in protecting us from infectious and other foreign invaders, such as grafts and certain tumors. Immunology and oncology thus have a long relationship, in evolutionary terms as well as within the biomedical sciences. The two fields have intersected again and again over the past half-century. We recount here some of these events, with a deliberate focus on T lymphocytes—from their discovery to their genetic engineering and use in cancer immunotherapy. Insightful observations into leukemogenesis in mice were at the root of the discovery of thymopoiesis and lymphocyte subsets. Subsequent observations led to the concept of immune surveillance, with eventual controversies on the role of immunity in tumor prevention and protection that were reconciled in the cancer immunoediting hypothesis. As progress was made in our fundamental understanding of antigen recognition, T cell activation, and T cell costimulation, translational researchers began to exploit the accumulating knowledge for cancer therapy. Tumor-infiltrating T cells were harnessed for adoptive cell therapy in a subset of melanoma patients; cancer vaccines were developed in an attempt to amplify endogenous tumor-specific T cell responses. Two of the most recent, most exciting therapeutic developments rest on the manipulation of costimulatory pathways governing T cell function. One, based on monoclonal antibody technology, enables the release of tumor-infiltrating T cells from inhibition mediated by costimulatory receptors such as CTLA-4 and PD-1, through either checkpoint blockade or depletion of regulatory T cells. The other, based on gene transfer technology, enables to repurpose patient’s T cells, targeting them to tumor antigens and augmenting their functional properties to overcome barriers erected by tumor cells and their microenvironment. These two forms of cancer immunotherapy were recognized by the Science magazine as the “breakthrough of the year” in 2013 () and by the US Food and Drug Administration, which approved the anti-CTLA-4 monoclonal antibody Ipilimumab in 2011 and granted breakthrough status to the CD19-specific chimeric antigen receptors (CARs) utilized at the University of Pennsylvania and Memorial Sloan Kettering Cancer Center for the treatment of pediatric and adult acute lymphoblastic leukemia in 2014. In this review, we aim to expose how basic discoveries in immunology led to these promising advances in cancer therapy.

In children with thymus aplasia, as in di George syndrome, thymus transplantation has reversed immunodeficiency (). The problem lies in donor availability and tissue compatibility. However, the recent spectacular success in creating a functional thymus organ by enforcing Foxn1 expression to reprogram mouse embryonic fibroblasts into fetal thymic epithelium () constitutes a first promising step in the provision of appropriate thymus tissue.

All of these results were initially regarded with some skepticism. Vague criticisms abounded, such as the one stating that mice must be unusual and that the results obtained would never be seen in humans. What needed to be checked, however, was that because the mice used had been raised in converted horse stables, the added trauma of neonatal thymectomy made them highly susceptible to infections. This criticism was soon quashed when it was shown that mice reared in a germfree facility, when thymectomized at birth did not develop wasting disease and yet were unable to reject skin grafts, even those that differed at the H-2 locus (). In 1968, the nude mouse was discovered (), and immunologists no longer had doubts about the immunological function of the thymus.

Although adult thymectomy had no effect on immunological capacity, it could conceivably have caused a problem in mice whose lymphoid system had been destroyed in some way, as for example by total-body irradiation. This proved to be the case in partially irradiated mice () and in lethally irradiated mice protected with bone marrow (). The use of adult thymectomized, heavily irradiated mice protected with bone marrow (ATxBM mice) proved invaluable for further elucidation of immune functions.

It seemed important to determine whether the thymus exerted its influence by seeding cells into the rest of the lymphoid system. Since no cell-surface markers had at that time been found to identify cells from different locations, use was made of the T6 strain of mice, the cells of which could be identified at metaphase by the presence of two minute chromosomes. Neonatally thymectomized F1 hybrid mice, in which one parent was T6, were grafted with thymus from the other parental strain and immunized with skin from various donors. An analysis of the chromosome constitution of the cells in metaphase in the spleen showed that 15% to 20% had originated from the thymus graft (). This suggested that the thymus did produce cells capable of migrating to the periphery and that presumably such recent thymus emigrants would have just matured before leaving or would mature in the peripheral lymphocyte pool to become fully competent lymphocytes.

Thymus grafting restored immune potential, and grafting of a foreign thymus induced specific tolerance to skin from the donor of the thymus graft (). This suggested that the thymus may be the seat where tolerance is learned: “Antigenic material might make contact with certain cell types differentiating in the thymus and in some way prevent these cells from maturing to a stage when they would be capable of reacting immunologically” (). This clear prediction of negative selection was proven some years later by using the so-called super antigens and transgenic mice, and both positive and negative selection of thymocytes were then worked out (). The major events in thymus cell differentiation have been summarized in a recent Timeline review ().

It was surmised that to induce a high percentage of leukemia in low-leukemic strains of mice, the virus had to be given neonatally because it needed to multiply in some cells present only in a newborn thymus. To test this hypothesis, the virus was given at birth but immediately after neonatal thymectomy, based on the prediction that thymus grafting performed later would not restore leukemogenesis. The neonatally thymectomized mice fared well until some weeks after weaning, when many became sick, wasted, and died. This had never been seen by anyone who had thymectomized adult mice (). Post-mortem examination revealed lesions in the liver suggestive of mouse hepatitis virus infection and marked diminution of lymphocytes in the blood and in the lymphoid tissues (). As lymphocytes were known to be involved in graft rejection and other immune responses, the neonatally thymectomized mice were tested for immunocompetence by grafting them with foreign skin before they had begun to show signs of wasting disease. Remarkably, they failed to reject foreign skin grafts, even when donors and recipients differed at the major histocompatibility locus, H-2 (). The neonatally thymectomized mice also lacked the ability to produce a normal antibody response to certain antigens, such as Salmonella typhi H antigen () and sheep erythrocytes ().

To obtain a high incidence of leukemia in mouse strains that were not highly prone to develop this malignancy, the virus had to be given at birth, and not later (). Thymectomy of virus-inoculated mice at 1 month of age prevented the disease (), and grafting a neonatal thymus as late as 6 months after adult thymectomy restored the potential for leukemia development (). Clearly, the virus must have remained latent, and indeed, it could be recovered from the non-leukemic tissues of thymectomized mice not grafted with thymus tissue ().

Prior to 1960, the thymus was thought to be a vestigial organ that had become redundant during evolution and was just a graveyard for dying lymphocytes. Even though recirculating small lymphocytes had been found by Gowans () to be immunocompetent cells able to initiate either cellular or humoral immune responses, thymus lymphocytes were deemed immunoincompetent since they did not recirculate, nor could they transfer immune responses to appropriate recipients. Furthermore, thymectomy, which had always been performed in adult animals, had no untoward effects on immune capacity. In 1959–1961, however, results obtained in a mouse model of lymphocytic leukemia induced by the Gross leukemia virus led to experiments using neonatally thymectomized mice.

The Identification of T and B Cells in Mice

Gowans et al., 1962 Gowans J.L.

McGREGOR D.D.

Cowen D.M. Initiation of immune responses by small lymphocytes. Miller et al., 1967 Miller J.F.

Mitchell G.F.

Weiss N.S. Cellular basis of the immunological defects in thymectomized mice. Miller and Mitchell, 1967 Miller J.F.

Mitchell G.F. The thymus and the precursors of antigen-reactive cells. Miller and Sprent, 1971 Miller J.F.

Sprent J. Thymus-derived cells in mouse thoracic duct lymph. As stated before, Gowans had clearly shown that recirculating small lymphocytes could respond both by a cellular immune response (as in skin graft rejection) and by producing antibody (). He considered that the same cell could take part in either, depending on the antigenic stimulus. As neonatally thymectomized mice were deficient in both cellular and at least some humoral responses, it was urgent to show that they had markedly reduced numbers of recirculating lymphocytes, not just blood lymphocytes. This was performed after cannulating the thoracic duct of neonatally thymectomized and ATxBM mice and collecting the lymph over a 24- to 48-hr period (). The conclusion was made that most thoracic duct lymphocytes (TDLs) in mice were thymus derived, and this was actually proven in subsequent experiments ().

Claman et al., 1966 Claman H.N.

Chaperon E.A.

Triplett R.F. Thymus-marrow cell combinations. Synergism in antibody production. Warner et al., 1962 Warner N.L.

Szenberg A.

Burnet F.M. The immunological role of different lymphoid organs in the chicken. I. Dissociation of immunological responsiveness. Burnet, 1962 Burnet F.M. The thymus gland. But were the same cells involved in antibody production and skin graft rejection? Two experimental systems suggested that this might not be the case. Claman () showed that irradiated mice given syngeneic bone marrow cells and syngeneic thymus cells could produce more antibody than when given either cell source alone. As no antibody markers were available at the time, the origin of the antibody-forming cells could not be easily identified. The situation was different for chickens in Burnet’s laboratory, where impairment of bursa function by testosterone injection caused the birds not to produce antibody, whereas thymus atrophy in sick birds prevented graft rejection (). Because mammals do not have a bursa, Burnet () surmised, “In mammals it is highly probable that the thymus also carries out the function of the bursa of Fabricius in the chicken, which is to feed into the body the cells whose descendants will produce antibody.”

Miller and Mitchell, 1967 Miller J.F.

Mitchell G.F. The thymus and the precursors of antigen-reactive cells. Miller and Mitchell, 1968 Miller J.F.

Mitchell G.F. Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes. Miller and Mitchell, 1969 Miller J.F.

Mitchell G.F. Thymus and antigen-reactive cells. Mitchell and Miller, 1968 Mitchell G.F.

Miller J.F. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. Figure 1 The Origins of T Cells and B Cells Show full caption Mitchell and Miller (1968) Mitchell G.F.

Miller J.F. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. Percent reduction of antibody-forming cells per spleen of adult thymectomized and heavily irradiated CBA protected with CBA bone marrow (ATxXBM), given (CBAXC57BL)F1 thoracic duct lymphocytes, and challenged with sheep erythrocytes. To the incubation mixture was added normal mouse serum (NMS; slightly toxic), C57BL anti-CBA serum (αCBA), or CBA anti-C57BL serum (αC57BL) as shown. The number of mice providing spleens in each group was three to six. Adapted from. These experiments demonstrated the existence of thymus-derived cells (later known as T cells) that were not antibody formers but were essential to allow cells derived from the bone marrow (later known as B cells) to produce antibody to certain antigens (later known as thymus-dependent antigens). In 1967 and 1968, Miller and Mitchell (), reconstituted neonatally thymectomized and adult thymectomized, irradiated CBA strain mice with CBA bone marrow and (CBAXC57BL)F1 TDLs and challenged them with sheep erythrocytes. The ATxBM mice in those experiments were injected with immunocompetent TDLs, and antibody-forming cells were produced that lysed sheep erythrocytes. This was shown by plaques when sheep red cells and spleen cells were layered onto agar plates. Now the time was appropriate to determine whether the TDLs gave rise to antibody-forming cells. It was done by simply adding to the plates antiserum against CBA strain antigens made by immunizing C57BL mice with CBA tissues or antiserum against C57BL made in CBA mice. The latter would be expected to kill any antibody-forming cells if they were derived from the (CBAXC57BL)F1 TDL and hence from the thymus-derived cells ( Figure 1 ). The results were spectacular, showing beyond any doubt that the antibody-forming cells did not originate from the TDLs, but rather from the bone marrow (). It proved that lymphocytes could be subdivided into two major groups: thymus-derived cells (later known as T cells) were not antibody formers but were essential to allow cells derived from the bone marrow (later known as B cells) to produce antibody in response to certain antigens (later known as thymus-dependent antigens). They were thus helper cells collaborating with other cell types, derived from the bone marrow, to enable these to produce antibody. The murine equivalent of the bursa was thus the bone marrow.

Good, 1969 Good R.A. Discussion after Miller, J.F.A.P. Gowans, 1969 Gowans J.L. Discussion after Miller, J.F.A.P. The existence of two major lymphocyte subsets was first regarded with some skepticism. At a meeting in Brooke Lodge (Augusta, Michigan) held in 1968, Good was “concerned at separating thymus-derived from marrow-derived cells” since the former “are in fact marrow derived-cells.” He also claimed to “have evidence that in the rabbit it (the bursa equivalent) resides in the ilial lymphoid tissue and in the lymphoid tissue of the appendix” (). At the same meeting, Gowans (), who had proven that recirculating small lymphocytes were immunocompetent, stated, “Had it not been for Dr Miller’s experiments I would have assumed that a single variety of small lymphocyte was involved in each of our experiments… If we have two cell lines that are collaborating, then we have specificity residing in two cell lines, one thymus-derived and the other marrow-derived. The problem is to bring these two specific cell lines together. Does this necessity for the two cells to find one another raise problems? It seems an inefficient mechanism if it rests only on chance contacts.” The then Professor of Immunology at the National University in Canberra, Australia, offered a less diplomatic critique and simply likened B and T cells to the first and last letter of the word “bullshit.”

The above somewhat simple experiments, performed without the use of modern technologies such as gene targeting, cluster of differentiation (CD) antigens, monoclonal antibody, and flow cytometry, changed the course of immunology. Thus, the existence of T and B cells required a reinvestigation of numerous immunological phenomena in terms of the role played by the two distinct cell types, including the carrier effect, immunological tolerance, immunological memory, immunodeficiency, autoimmunity, and genetically determined immune responsiveness. An avalanche of work soon followed as investigations were focused on defining and investigating further lymphocyte subsets and their function.

Steinman and Cohn, 1973 Steinman R.M.

Cohn Z.A. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. Zinkernagel and Doherty, 1974 Zinkernagel R.M.

Doherty P.C. Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Townsend et al., 1986 Townsend A.R.M.

Rothbard J.

Gotch F.M.

Bahadur G.

Wraith D.

McMichael A.J. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. H genes would make their way into T cells, after all (see below). Since T and B cells utilize different molecules as antigen-specific receptors, do they perceive antigen in different ways? This is indeed the case: whereas B cells can bind soluble antigen, T cells generally recognize antigen only if displayed on the surfaces of antigen-presenting cells (APCs), such as dendritic cells () or virus-infected cells. Cytotoxic T cells isolated from mice recovering from a virus infection were tested for their capacity to kill virus-infected target cells in vitro. Killing was observed but only if the target cells had the same major histocompatibility complex (MHC) haplotype as the mice from which the T cells were obtained (). This suggested that there might be an association between a virus product and MHC molecules at the cell surface and that the specificity of the TCR was directed to both MHC molecules and virus-encoded products. The phenomenon became known as MHC restriction and the MHC molecules involved as restriction elements. T cells in fact recognize relatively short peptide fragments () that become wedged in the jaws of the MHC molecules. How T and B cells perceive antigen is highly relevant for tumor immunity and the escape of immunogenic tumor cells from any host immune response. Loss of cell-surface antigen or MHC molecules from genetically unstable tumor variants would of course prejudice or prevent the response of any specific B and T cells. CARs would later capitalize on the respective advantages of both targeting modalities—and Ig Vgenes would make their way into T cells, after all (see below).