Naive T-cell Expansion, Differentiation, Contraction and Memory T-cell Generation
Whereas cytoreductive anti-tumor therapies can reduce disease burden, residual tumor cells must be eradicated or held in check by the immune system. The most compelling evidence for a T-cell mediated immune mediated anti-tumor response can be derived from the adoptive transfer of allogeneic donor lymphocytes used to treat leukemia recurrence following transplantation105
. Assays for minimal residual disease in CML have shown a gradual reduction in tumor burden, often requiring months to years to achieve the full biological effect. These data are consistent with a critical threshold number of anti-tumor reactive T cells that is needed to achieve and sustain an anti-leukemia effect.
cells can contribute to anti-tumor responses. With age and repetitive radiation or chemotherapy courses, fewer näive T-cells are produced106
. Under those conditions, TM
cells may be the dominant anti-tumor reactive T cell population. Naive T cells encountering immunogeneic tumor peptides or antigens can be skewed toward an anti-leukemia response, especially during periods of lymphopenia induced by cytoreductive therapies. This results in cytokine accumulation (e.g. IL-7, IL-15) and substantial T-cell homeostatic expansion and differentiation of naive T cells into TE
cells that reduce tumor burden107
. Typically within the first week, a second and profound contraction phase occurs due to activation-induced cell death, cytokine consumption, and/or inhibitory mechanisms (e.g. exposure to IFN-gamma or TGFβ immunosuppressive cytokines, upregulation of CTLA-4 or PD-1 inhibitory coreceptors or TRAIL or Fas pathway death receptors). Following the contraction phase, a small subset of antigen-reactive TEM
, and putative memory stem cells survive, each of which may retain an anti-tumor response capacity. In the presence of CD4+
T-cell help, post-homeostatic proliferation-induced memory-like cells are produced which can have similar responses to conventional CD8+
The precise mechanisms by which CD4+
T cells maintain CD8+
T cell memory are still poorly understood and this may impact the efficacy of CD8+
T cell transfer.
If tumor antigen levels are sufficient to trigger their TCR, memory cells may receive survival signals that promote their persistence and function, thereby permitting further reductions in tumor burden and continued immune surveillance. If antigen levels are too high, T cells may become exhausted and are known to express inhibitory molecules such as PD-1, which upon engagement by PD-1 ligand (PD-L) render antigen-reactive T cells hyporesponsive in terms of proliferation, cytokine production, and killing capacity20
. If antigen levels are too low, these memory populations would become quiescent until they are re-engaged by antigens that trigger their TCR and sufficient adjuvant signals such as cytokines or costimulatory pathway receptors that can re-awaken cells to proliferate and lyse tumor cells.
The Tumor Microenvironment
A key factor that regulates the size and activation status of the memory cell pool is tissue location. Anti-tumor reactive TM cells may reside in secondary lymphoid organs, parenchymal organs, bone marrow, or the tumor microenvironment itself. Some but not all of these environments are conducive to an anti-tumor response by providing positive signals (e.g. costimulation, stimulatory cytokines) and only a limited number of inhibitory signals or cell populations that restrain memory cell activation, expansion or acquisition of lytic function. Another key factor in the naive and memory T-cell response is the capacity of anti-tumor reactive T cell to migrate to the tumor site. Therefore, T-cell populations not residing at the tumor site and lacking necessary addressins, selectins or chemokine receptors for migration to tumor site are unlikely to substantively contribute to the immune response.
The immune environment in which TN
cells reside may suppress the anti-tumor immune response by affecting T-cell activation, proliferation, pro- or anti-inflammatory cytokine expression, cytolytic molecule expression, pro- or anti-apoptotic molecule expression or the regulation of receptors involved in migration. The size and activation status of the TM
cell compartment is determined by the balance between positive and negative signals delivered to the memory T-cell. The positive and negative immune pathways work in concert but suppression tends to trump over stimulation. The lack of the stimulatory factors may also predispose the immune cells to the suppressive pathways. These suppressive pathways may predominate even more in the aged individual which may significantly limit immune interventions in this population. Typically, the tumor highjacks its microenvironment to foster tumor growth and discourage a productive immune response. Tumor cells such as those seen in Hodgkin's lymphoma cells can produce immunosuppressive cytokines such as IL-10 or TGFβ that suppress T-cell proliferation or induce Tregs that constrain TE
-cell responses in the tumor microenvironment109
. Tumor cells can actively attack the attackers by expressing fasL and PD-L110
Tumor cells may express high levels of the intracellular tryptophan catabolic pathway, indoleamine 2,3 dioxygenase (IDO), as has been demonstrated for AML cells111
In addition, CD11c+
plasmacytoid dendritic cells (pDC) present in draining lymph nodes or the tumor microenvironment of rodents and humans can express high levels of IDO, which may be upregulated by tumor cells (e.g. melanoma, breast cancer), Toll-like receptor-9 (TLR-9) ligation from exposure to bacteria products, or the T-cell response itself via the elaboration of IFN-gamma or surface expression of CTLA-4112
. High IDO levels act in the local microenvironment to stimulate integrated stress response pathways in T cells activated by amino acid starvation resulting in cell cycle arrest or apoptosis. In rodents, IDO expression in pDC has been shown to augment mature Tregs and upon TLR-9 ligation blocks the conversion of Tregs into Th17-like cells113
, while in humans, TLR-9 activated pDC can convert naive T cells into Tregs114
Tumors can cause T-cell dysfunction by altering TCR signaling mechanisms115
. Depletion of the amino acid L-arginine by cytoplasmic arginase I profoundly inhibits CD4+
. L-arginine depletion is associated with the down-regulation of the TCR zeta chain in T cells that infiltrate in the tumor microenvironment. Arginase I is expressed at high levels in myeloid-derived suppressor cells (MDSC) that infiltrate the tumor microenvironment116
. Murine MDSC have been shown to have an increased uptake of L-arginine due to high levels of a cationic amino acid transporter, 2B117
. In addition, prostaglandin-E2 is synthesized by MDSC, which most likely contributes to immune suppression in the tumor microenvironment118
. In humans, MDSC are CD34+
, indicative of a myeloid lineage119
. In mice, Gr-1low
cell fractions possessed suppressive potential119
. The latter cells co-expressed F4/80, high levels of Ly6C, and low levels of CSF-1 receptor (CD115). Suppression was observed, albeit at as lesser potency on a cell-by-cell basis for Gr-1hi
cells. IFN-gamma or lipopolysaccharide not only activates MDSC, but suppresses the differentiation of DC from bone marrow myeloid progenitors. Therefore, an inflammatory response to tumor cells can result in the generation of MDSC in the tumor microenvironment, offsetting the host anti-tumor immune response. Intriguingly, in patients with renal cell carcinoma, IL-2 therapy markedly increased MDSC percentages and arginase I levels120
. Thus, IL-2 has a dual effect on the immune response by increasing TE
cells and NK cells as well as Tregs and MDSC. MDSC also can suppress T-cell function via other mechanisms such as the elaboration of nitric oxide or regulation of CD80 and PD-L expression. Vascular endothelial cell growth factor (VEGF), known to be produced by almost all tumor cells, has been shown to inhibit the development of DC from the bone marrow, which was associated with an increase in immature, immune suppressive Gr1+
. In some types of cancer patients, immature DC in fact are increased in the peripheral blood of patients.
A functional state of tumor-mediated suppression may occur as a result of deviation of the immune response toward a non-favorable TE cell phenotype (e.g. Tregs type I; anti-inflammatory cytokine producing Th2 cells; TGFβ-secreting Th3 cells). Alternatively, tumor cells may inhibit antigen recognition by a variety of mechanisms. For example, some tumors down-regulate major histocompatibility complex antigens (e.g. MHC class I in neuroblastoma cells) or potentially immunodominant antigens (e.g. EBV nuclear antigens, 3A, B, C in Hodgkin's disease). Tumor cells can also secrete “decoy” molecules such as soluble MICA which can inhibit NKG2D-mediated killing by NK cells. Defective APC function has been reported in cancer patients as a consequence of low expression of costimulatory molecules (e.g. B7 ligands), exposure to high levels of IL-10 that result in tolerogenic DC, or high expression of inhibitory co-receptors (e.g. PD-L).
Therapeutic Strategies to Overcome Immune Contraction and Suppression
Bypassing activation-induced cell death (AICD) is critical for sustaining activated immune cell engraftment and function. However, many cytokines and pathways can exert opposing effects. IFN-gamma is a major mediator that is critical for many anti-tumor effector functions as well as a major mediator limiting T-cell responses, particularly CD4+
T cells via AICD122
. During T-cell activation, CD25 (IL-2Rα chain) is upregulated followed by the down-regulation of CD127 (IL-7Rα chain) at the time of TE
cell to effector/memory transition. As such, it is reasonable to consider the use of IL-2 or IL-7 to expand TE
cells. However, the biology of IL-7 and other cytokines such as IL-15 is complicated due to the effects of presentation of the cytokine by DC's potentially yielding opposing effects on the T cell123
. Moreover, the consumption of cytokines that can occur during the expansion phase, particularly following lymphopenia and homeostatic expansion, may result in a relative deficiency of cytokines to support activated TE
cells. Exogenous IL-2 administration has been shown to increase TE
cells as well as NK cells and Tregs. Of note, high doses of IL-2 can cause AICD of activated T cells, and IL-2 withdrawal can cause cytokine-deprivation induced apoptosis. Therefore the available levels of IL-2 for IL-2R engagement are critical in determining the ultimate T cell fate. Recent data suggest that the binding of IL-2 with an antibody can result in supraphysiologic effects in vivo and even bypass the need of NK cells for IL-15124,125
. In rodents, IL-7, the second member of the IL-2R common gamma chain family of cytokines, has been shown to be a critical growth factor for CD8+
cell survival. In humans, exogenous IL-7 supported a sustained increase in peripheral blood CD4+
T cells with broadening of the TCR repertoire diversity, induced T cell cycling and anti-apoptotic protein (bcl2) upregulation, and expanded TN
cells, with a proportional diminution in Treg frequency126
. Since IL-7 was well-tolerated in this first in-human study, IL-7 administration may prove especially useful in situations in which IL-7 levels are limiting such as following homeostatic expansion during the resolving lymphopenia phase.
IL-15, the third member of the IL-2R common gamma chain family of cytokines, has been shown to be essential for memory cell survival. In vivo studies in rodent and non-human primates have demonstrated the capacity of IL-15 administration to facilitate T-cell survival, especially antigen-specific central memory T cells. Studies of human T cells indicate that IL-15 favors the in vitro
generation of TCM
cells and also appears to be critical for NK cell survival. IL-15 is being readied for first-in-human testing in the near future. However, the “trans” presentation that IL-15 appears to require for optimal effects suggests that simple administration of the cytokine may not be sufficient127
. IL-21, the fourth member of this family that is closely related to IL-2, also promotes the function of CD8+
. In contrast to IL-2, IL-21 inhibits the maturation of CD8+
cells into granzyme B- and CD44-expressing effector CD8+
. At the same time, IL-21 increased the anti-tumor regressive capacity of adoptively transferred CTL. These data are consistent with the paradigm that cytokines, such as IL-21, which limit both the stage of TE
cell differentiation and preserve the expression of key cell surface homing receptors such as L-selectin. Thus, IL-7, IL-15 and IL-21 are new candidate cytokines that may favor the persistence of TM
cells with improved anti-tumor properties, as compared to IL-2. New approaches for cytokine delivery, such as cytokine-anti-cytokine antibody complexes may provide a superior methodology to achieve more pronounced in vivo biological effects by increasing their otherwise short serum half-life.
Thus far, the success of bypassing negative regulators (e.g. CTLA-4 or PD-1 pathway blockade for T cells, killer inhibitory receptor blockade for NK cells) may result in augmented anti-tumor effects but may also result in possible auto-reactive toxicities. For example, antibodies directed against CTLA-4 have shown promising results in clinical trials in augmenting endogenous anti-tumor reactive T cells while at the same time inducing autoimmune-mediated destruction of non-tumor cells in some patients130
. Anti-PD-1 and anti-PD-L1 antibodies are in clinical trials in cancer patients; anti-PD-L1 antibody may be particularly useful in preventing the T-cell exhaustion state that chronic antigen activation may cause20
. However, in vivo
studies have suggested that targeting multiple members of the PD1/PD-L family may be needed for optimal effects. Agonistic antibodies against costimulatory pathway members (e.g. CD40/CD40L, OX40/OX40L, 4-1BB/4-1BBL) alone or combined with blockade of inhibitory co-receptors may prove to be particularly efficacious in patients with both APC and T-cell dysfunction.
A chemical inhibitor of the IDO pathway (1-methyl-tryptophan) is being studied in phase I trials in cancer patients. COX-2 inhibitors have been used successfully, especially in patients with solid tumors, to reverse PGE2-mediated suppression and the frequency of inducible Tregs that can occur as a result of the tumor microenvironment131
. MDSC-mediated inhibition of anti-tumor T-cell or NK cell immune responses may be overcome by sunitinib malate132
, a receptor kinase inhibitor, TGFβ neutralization133
, modulators of arginine metabolism (see above) or nitric-oxide synthase inhibitor [e.g. N(G) nitro-L-arginine methyl ester (L-NAME)]134
. Anti-VEGF antibody in rodent but not human studies decreases suppressive DC numbers120,121
Cellular depletion approaches such as those targeting Tregs (e.g. IL-2 diphtheria toxin, a.k.a. denileukin diftitox or Ontak®) have been variably efficacious in augmenting endogenous T-cell responses by depleting Tregs. The transfer of ex vivo
expanded and activated anti-tumor reactive T cells, including those that are forced to express an anti-tumor reactive TCR by gene transfer, have been reported to be efficacious in patients with melanoma, generally an immune responsive disease135
. Rodent studies have indicated that combined in vivo Treg depletion with the adoptive transfer of syngeneic CTL136
may be a useful strategy that circumvents the in vivo dysfunction of anti-tumor reactive T-cells (e.g. TCR zeta chain down-modulation) and inhibits the suppression conferred by Tregs.
The role of CD4+
T cells and the ratio of their subsets may be critical in maintaining CD8+
T-cell responses (survival and function) also needs to be explored. More attempts at combination approaches need to be undertaken. Recent data also suggests that activated DC may do more than simply initiate the adaptive response but may also play a role in maintaining antigen-specific CD8+
T-cell function and survival137
. These data indicate that cytoreductive conditioning in HSCT may do much more than simply promote T-cell expansion via homeostasis but instead activates DC via TLR. Understanding the mechanisms involved may have a profound impact on the use of HSCT and adoptive immunotherapy. Finally, it has been shown in numerous models that peripheral immune readouts may have no bearing on anti-tumor responses. There needs to be studies that understand how normal contraction processes occur within any attempt to stimulate or provide immune responses. AICD coupled with immune suppression and deprivation of supportive cells and cytokines may prematurely diminish responses and make the host resistant to further attempts at stimulation. Targeting immune responses to the tumor site and, importantly, assessing immune effects within the tumor itself may offer the best indicators of success as well as limit systemic toxicities. Understanding normal immune contractions/suppression and how the tumor can further subvert it is critical if the immune system is to be applied in cancer therapies.