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Recent preclinical studies suggest that vaccination following adoptive transfer of CD8+ T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8+ T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8–16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp10025–33 melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8+ T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.

Enhanced effector activity such as cytokine secretion, cell proliferation , cytolytic activity, etc. in response to in vitro stimulation has been widely used as a means to predict in vivo activity after immune mediated interventions, such as adoptive transfer or systemic vaccination. Contrary to this, here we show that the enhanced in vivo survival and anti-tumor activity of cytokine conditioned early activated CD8+ T cells inversely correlates with their effector functions in vitro. When compared with late effector PmelIL-12, early effector showed decreased production of IFNγ and TNFα, and decreased expression of the degranulation marker CD107a in response to in vitro reactivation. However, after transfer into lymphodepleted hosts, early effector PmelIL-12 survived for at least 20 days and were able to effectively control the progression of B16 tumors in a superior manner than late effector PmelIL-12. Thus, effector activity in response to ex vivo antigenic restimulation does not necessarily predict in vivo activity. Other parameters such as surface expression of functional markers (CCR7, CD62L) and /or survival (CD127) could serve as better predictors of in vivo activity. Developing and validating accurate predictors of in vivo lymphocyte function would greatly facilitate our ability to rationally design novel immune based therapies for clinical trials that have significant anti-tumor activity.

It has been recently shown that early effector CD8+T cells are more efficient at killing tumors than late effector CD8+T cells. Since expression of CD62L is progressively lost as T cells differentiate, its expression on activated CD8+T cells has been associated with superior antitumor activity. In our study, we have shown that in vitro priming in the presence of IL-12 results in the accumulation of CD62LhiCD8+ T cells. The antiapoptotic effects of IL-12 were found to be selective for this population and was associated with downregulation of caspases 3 and 8, Bax, and Bad. We also found that IL-12 modulates the expression of CD62L. OT-1 cells primed with SIINFEKL with or without IL-12 were sorted into CD62Llo and CD62Lhi populations three days after priming and recultured with or without IL-12. Exposure of untreated CD62LloCD8+cells to IL-12 after sorting resulted in increased CD62L levels. CD62LloCD8+cells primed in the presence of IL-12 transiently upregulated CD62L expression. However, the levels decreased by day 7 unless cells were re-exposed to IL-12. The fact that effects were seen on sorted populations, suggests that IL-12 has a direct effect on the expression of CD62L. Finally, we found that CD8+CD62Lhi cells generated after priming with IL-12 had increased in vivo survival and enhanced recall capabilities. When compared with CD62Lhi cells primed with antigen alone, IL-12-treated- CD62Lhi cells showed enhanced anti-tumor capabilities. In conclusion, our data supports the findings that increased expression of CD62L on effector T cells results in enhanced anti-tumor activity, and suggests IL-12 treatment as an effective strategy for the generation and maintenance of such subpopulation.

Tumor-associated immune suppression is known to be an important contributor to cancer progression. One such mechanism is the abnormal accumulation of ImC in the peripheral blood. Although both tumor burden and CTX-induced accumulation of ImC are well described in animal studies, human studies have been limited. Thus, the goal of our study was to determine whether levels of ImC in whole blood correlate with clinical cancer stage in cancer patients (pts). Flow cytometry analyses were performed on whole peripheral blood from newly diagnosed solid tumor pts. ImC population was defined as lineage−CD33+HLA-DR−CD11b+. Peripheral blood specimens from 14 normal healthy controls and 35 cancer pts (stages: I, n=3; II, n=9; III, n=9; IV, n=14), revealed greater ImC in cancer pts vs. normal (3.21% vs. 1.47%; p=.002). Stage IV pts had the highest percentage of ImC (4.39%). Initial results from stage II-III breast cancer pts (n=10) treated with preoperative chemotherapy showed that CTX treatment was associated with significant increases in circulating ImC compared with baseline (2.91% vs. 10.57%; p=0.001), and also when compared to taxane chemotherapy independent of G-CSF (10.57% vs. 3.25%; p=0.00025). In conclusion, overall ImC levels correlate with clinical cancer stage and CTX treatment leads to transient increases in ImC in breast cancer pts.

Regulatory T cells inhibit immune responses mediated by T cells. Increased circulating levels of T-regs in patients (pts) have been reported in some solid tumors. Moreover, increased apoptosis among circulating CD8+ T cells has been reported in cancer pts. The goal of our study was therefore to determine how levels of circulating T-regs and CD8+T cells correlate with clinical cancer stage in a diverse cohort of pts. Flow cytometry analyses were performed on whole blood from 14 normal healthy controls and 35 cancer pts with newly diagnosed tumors prior to any therapy (stages: I/II, n=12; III/IV n=23). Although no significant difference was observed in T-regs between normal and stage I/II cancer pts (4.36% vs. 4.92%), levels were significantly increased in stage III/IV pts (7.26%; p=0.039). Moreover, there was an inverse correlation between CD8+T cell frequencies and clinical cancer stage, with stage III/IV pts having the lowest levels relative to normal (3.97% vs. 7.01%; p=0.036). In conclusion, T-regs were increased only in pts with advanced stage III/IV cancer and percentage of total CD8+T cells inversely correlated with clinical tumor stage. Thus, increased levels of T-regs and decreased levels of CD8+ T cells might contribute to cancer-mediated immunosupression.