Purpose: The Vδ1+ subset of γδ T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vδ1+ T-cell–based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation. Experimental design: We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2–3 weeks) Vδ1+ T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL). Results: We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinical-grade cell culture bags) and differentiation of cytotoxic Vδ1+ (DOT) cells. These expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro. When transferred in vivo, DOT cells infiltrated tumors and peripheral organs, and persisted until the end of the analysis without showing signs of loss of function; indeed, DOT cells proliferated and produced abundant IFNγ and TNFα, but importantly no IL17, in vivo. Critically, DOT cells were capable of inhibiting tumor growth and preventing dissemination in xenograft models of CLL. Conclusions: We provide a clinical-grade method and the preclinical proof of principle for application of a new cellular product, DOT cells, in adoptive immunotherapy of CLL. Clin Cancer Res; 22(23); 5795–804. ©2016 AACR.

We developed a novel experimental strategy to study T cell regeneration after bone marrow transplantation. We assessed the fraction of competent precursors required to repopulate the thymus and quantified the relationship between the size of the different T cell compartments during T cell maturation in the thymus. The contribution of the thymus to the establishment and maintenance of the peripheral T cell pools was also quantified. We found that the degree of thymus restoration is determined by the availability of competent precursors and that the number of double-positive thymus cells is not under homeostatic control. In contrast, the sizes of the peripheral CD4 and CD8 T cell pools are largely independent of the number of precursors and of the number of thymus cells. Peripheral “homeostatic” proliferation and increased export and/or survival of recent thymus emigrants compensate for reduced T cell production in the thymus. In spite of these reparatory processes, mice with a reduced number of mature T cells in the thymus have an increased probability of peripheral T cell deficiency, mainly in the naive compartment.

To fulfill its mission, the immune system must maintain a complete set of different cellular subpopulations that play specific roles in immune responses. We have investigated the mechanisms regulating CD4⁺CD25⁺ regulatory T (Treg) cell homeostasis. We show that the expression of the high-affinity IL-2Rα endows these cells with the capacity to explore the IL-2 resource, ensuring their presence while keeping their number tied to the number of CD4⁺ T cells that produce IL-2. We show that such a homeostatic mechanism allows the increased expansion of T cells without causing disease. The indexing of Treg cells to the number of activated IL-2-producing cells may constitute a feedback mechanism that controls T cell expansion during immune responses, thus preventing autoimmune or lymphoproliferative diseases. The present study highlights that maintenance of proportions between different lymphocyte subsets may also be critical for the immune system and are under strict homeostatic control.

Homeostasis of lymphocyte numbers is believed to be due to competition between cellular populations for a common niche of restricted size, defined by the combination of interactions and trophic factors required for cell survival. Here we propose a new mechanism: homeostasis of lymphocyte numbers could also be achieved by the ability of lymphocytes to perceive the density of their own populations. Such a mechanism would be reminiscent of the primordial quorum-sensing systems used by bacteria, in which some bacteria sense the accumulation of bacterial metabolites secreted by other elements of the population, allowing them to “count” the number of cells present and adapt their growth accordingly. We propose that homeostasis of CD4⁺ T cell numbers may occur via a quorum-sensing-like mechanism, where IL-2 is produced by activated CD4⁺ T cells and sensed by a population of CD4⁺ Treg cells that expresses the high-affinity IL-2Rα-chain and can regulate the number of activated IL-2-producing CD4⁺ T cells and the total CD4⁺ T cell population. In other words, CD4⁺ T cell populations can restrain their growth by monitoring the number of activated cells, thus preventing uncontrolled lymphocyte proliferation during immune responses. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled T cell activation and autoimmunity. Finally, we present a mathematical model that describes the key role of IL-2 and quorum-sensing mechanisms in CD4⁺ T cell homeostasis during an immune response.