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CD4+FoxP3+regulatory T cells (Tregs) have been shown to suppress T cell–mediated host immune responses against self- and nonself-antigens; however, the impact of CD4+Tregs on human antitumor immune responses and their influence on cancer treatment are unknown. In the present study, we explored the factors that influence CD4+Treg reconstitution in patients receiving adoptive immunotherapy following conditioning regimens designed to enhance T-cell function and evaluated potential associations between CD4+Treg levels and clinical responses to therapy. The analysis of 4 trials employing nonmyeloablative chemotherapy with or without total body irradiation (TBI) before adoptive T-cell transfer revealed that the percentage and number of reconstituting CD4+FoxP3+Tregs observed in the peripheral blood was higher in nonresponders than in responders. The addition of TBI resulted in a further depletion of CD4+Tregs, and the degree of depletion was dependent on the TBI dose. The number of administered doses of IL-2 was found to be positively associated with peripheral Treg reconstitution. These observations provide strong evidence that endogenous CD4+Tregs have a negative impact on cancer therapy, and suggest that strategies reducing Treg levels may provide clinical benefit to cancer patients. All 5 clinical trials are registered at www.clinicaltrials.govas NCT00001832, NCT00096382, NCT00335127, NCT00509496, and NCT00513604.

Cluster of differentiation (CD)8+T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8+T cells for patient treatments is controversial and understudied. We investigated human CD8+T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or “exhaustion,” maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.

IL-2 and IL-21 are closely related cytokines that might have arisen by gene duplication. Both cytokines promote the function of effector CD8+T cells, but their distinct effects on antigen-driven differentiation of naive CD8+T cells into effector CD8+T cells are not clearly understood. We found that antigen-induced expression of Eomesodermin (Eomes) and maturation of naive CD8+T cells into granzyme B- and CD44-expressing effector CD8+T cells was enhanced by IL-2, but, unexpectedly, suppressed by IL-21. Furthermore, IL-21 repressed expression of IL-2Ra and inhibited IL-2–mediated acquisition of a cytolytic CD8+T-cell phenotype. Despite its inhibitory effects, IL-21 did not induce anergy, but instead potently enhanced the capacity of cells to mediate tumor regression upon adoptive transfer. In contrast, IL-2 impaired the subsequent antitumor function of transferred cells. Gene expression studies revealed a distinct IL-21 program that was characterized phenotypically by increased expression of L-selectin and functionally by enhanced antitumor immunity that was not reversed by secondary in vitro stimulation with antigen and IL-2. Thus, the efficacy of CD8+T cells for adoptive immunotherapy can be influenced by opposing differentiation programs conferred by IL-2 and IL-21, a finding with important implications for the development of cellular cancer therapies.

Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation.

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) maintains peripheral tolerance by suppressing T-cell activation and proliferation but its precise role in vivo remains unclear. We sought to elucidate the impact of CTLA-4 expression on self/tumor-reactive CD8+T cells by using the glycoprotein (gp) 100–specific T-cell receptor (TCR) transgenic mouse, pmel-1. pmel-1 CLTA-4–/–mice developed profound, accelerated autoimmune vitiligo. This enhanced autoimmunity was associated with a small but highly activated CD8+T-cell population and large numbers of CD4+T cells not expressing the transgenic TCR. Adoptive transfer of pmel-1 CLTA-4–/–CD8+T cells did not mediate superior antitumor immunity in the settings of either large established tumors or tumor challenge, suggesting that the mere absence of CTLA-4–mediated inhibition on CD8+T cells did not directly promote enhancement of their effector functions. Removal of CD4+T cells by crossing the pmel-1 CLTA-4–/–mouse onto a Rag-1–/–background resulted in the complete abrogation of CD8+T-cell activation and autoimmune manifestations. The effects of CD4+CLTA-4–/–T cells were dependent on the absence of CTLA-4 on CD8+T cells. These results indicated that CD8+CLTA-4–/–T-cell–mediated autoimmunity and tumor immunity required CD4+T cells in which the function was dysregulated by the absence of CTLA-4–mediated negative costimulation.

Interleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hiT cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hiT cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hiT cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hiFoxp3+regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8+T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27LoCD28LoCD45RA-CD62 ligand-(CD62L-) CC chemokine receptor 7-(CCR7-) interleukin-7 receptor αLo(IL-7RαLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+CD28+tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+CD28+CD62L-CCR7-profile, which may explain in part their ability to mediate tumor destruction. (Blood. 2005;105:241-250)

Tumor infiltrating lymphocytes (TIL) were cultured from 17 B-cell lymphoma specimens derived from patients with predominantly low-grade malignancies. Specimens included 15 lymph-node biopsies, 1 malignant pleural effusion, and PBL from 1 patient with circulating lymphoma cells. The phenotypic and proliferative characteristics of TIL cultured in interleukin-2 (IL-2) were studied, as well as cytolysis and cytokine secretion in response to autologous tumor. Flow cytometry of fresh tumor suspensions showed that 50% of cells (median) were malignant B cells and 36% were infiltrating T lymphocytes. After culture for approximately 1 month, TIL were 75% ± 8% CD3+(mean ± SEM), 47% ± 8% CD4+and 35% ± 7% CD8+. TIL proliferation was modest in most cases: the median maximum expansion was 32-fold in 25 days. Lysis of autologous tumor in 4-hour 51Cr release assays was mediated by 2 of 12 TIL studied, but was nonspecific. However, these same two TIL, when cocultured with various tumor stimulators, preferentially secreted tumor necrosis factor-α and granulocyte-macrophage colony-stimulating factor after autologous tumor stimulation; unstimulated TIL secreted undetectable or barely detectable levels of these cytokines. In one TIL culture, cytokines were secreted by purified CD4+TIL but not by CD8+cells, and secretion was completely abrogated by the anti-major histocompatibility complex (MHC) class II antibody IVA12. Thus, although specific cytokine secretion by lymphoma TIL in response to autologous tumor was observed, it occurred in fewer than 20% of patients studied.

Immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells generated from autologous lymphocytes has produced significant tumor regressions in patients with advanced cancer. In the current study, we reviewed the hematologic effects associated with this therapy in our initial 42 patients. Eighty-eight percent of the treated patients developed anemia that required ≥4 units of red cell transfusions, and 43% received at least 8 units. Only a blood loss of 2 to 3 units could be attributed to repeated phlebotomy, cytophereses, and hemodilution. IL-2 administration also resulted in thrombocytopenia as well as lymphopenia and eosinophilia. Forty-three percent of patients developed platelet counts of ≤50,000/μL, and 36% of the total group required platelet transfusions. Mild neutropenia and a rebound lymphocytosis followed discontinuation of IL-2 treatment. To explore the possible mechanisms for these hematologic effects, standard hematopoietic colony assays were conducted on serial blood samples from five patients. IL-2 produced a significant decline in circulating erythroid (BFU-E) and granulocytic/ macrophage (CFU-C) progenitors, which rebounded after the discontinuation of IL-2 therapy. Infusion of IL-2 also resulted in measurable serum levels of γ-interferon. Some of the hematologic effects of immunotherapy with LAK cells and IL-2 may be the result of IL-2-mediated suppression of hematopoiesis.© 1987 byGrune & Stratton, Inc. 0006-4971/87/6906-0017$3.00/0

Both congenital and acquired antithrombin-lll (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human α 1-antitrypsin (a,AT) cDNA in yeast. Alteration of the α 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-lll. Alteration of two further residues, to give a five-residue sequence identical to AT-lll, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglyco-sylated and have shorter in vivo half-lives (t1/2) than human a, AT. These antithrombins should be suitable for therapeutic replacement of AT-lll in cases of congenital deficiency and in conditions associated with acquired AT-lll deficiency, such as disseminated intravascular coagulation.