In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization: implications for adoptive immunotherapy

Abstract

The development of T cell tolerance directed toward tumor-associated Ags can limit the repertoire of functional tumor-reactive T cells, thus impairing the ability of vaccines to elicit effective antitumor immunity. Adoptive immunotherapy strategies using ex vivo expanded tumor-reactive effector T cells can bypass this problem; however, the susceptibility of effector T cells to undergoing tolerization suggests that tolerance might also negatively impact adoptive immunotherapy. Nonetheless, adoptive immunotherapy strategies can be effective, particularly those utilizing the drug cyclophosphamide (CY) and/or exogenous IL-2. In the current study, we used a TCR-transgenic mouse adoptive transfer system to assess whether CY plus IL-2 treatment rescues effector CD4 cell function in the face of tolerizing Ag (i.e., cognate parenchymal self-Ag). CY plus IL-2 treatment not only enhances proliferation and accumulation of effector CD4 cells, but also preserves the ability of these cells to express the effector cytokine IFN-gamma (and to a lesser extent TNF-alpha) in proportion to the level of parenchymal self-Ag expression. When administered individually, CY but not IL-2 can markedly impede tolerization, although their combination is the most effective. Although effector CD4 cells in CY plus IL-2-treated self-Ag-expressing mice eventually succumb to tolerization, this delay results in an increased level of in situ IFN-gamma expression in cognate Ag-expressing parenchymal tissues as well as death via a mechanism that requires direct parenchymal Ag presentation. These results suggest that one potential mechanism by which CY and IL-2 augment adoptive immunotherapy strategies to treat cancer is by impeding the tolerization of tumor-reactive effector T cells.

FIGURE 1

CY and IL-2 treatment impedes self-Ag-induced effector CD4 cell tolerization. A total of 5 × 10⁵ resting effector clonotypic CD4 cells was relabeled with CFSE and adoptively retransferred into the indicated secondary adoptive transfer recipients that were either nontreated (−), treated daily with 10⁴ U of IL-2 (2), treated once the day before adoptive transfer with 180 mg/kg CY (C) or with CY plus IL-2 (C2). Four days after retransfer, the clonotypic CD4 cells were recovered from spleens for analysis. A, Representative histograms showing CFSE dilution (i.e., proliferative response), with a dotted line placed directly to the left of undivided cells. B, Frequencies of clonotypic CD4 cells. C, Representative histograms showing clonotypic CD4 cell intracellular IFN-γ and TNF-α expression following in vitro restimulation with HA peptide-pulsed APCs for untreated and CY plus IL-2-treated recipients. The percentage of clonotypic CD4 cells expressing cytokines as well as the level of cytokine expression (mean fluorescence (MF)) are shown. The cutoff for positive cytokine express was determined using an isotype control Ab (data not shown). D and E, Total IFN-γ and TNF-α expression, respectively, is shown for the indicated adoptive retransfer recipient groups in comparison to the primary effectors before retransfer (1^o). B–D, n = 4 for C3-HA^{low and high} recipients groups, and n = 3 for NT and NT plus vacc-HA recipient groups.

FIGURE 2

CY and IL-2 treatment impedes self-Ag-induced naive CD4 cell tolerization. A total of 2.5 × 10⁶ naive CFSE-labeled clonotypic CD4 cells was adoptively transferred into the indicated recipients that were either treated with CY plus IL-2 or not treated and recovered from spleens 4 days posttransfer. A, Representative CFSE dilution profiles. B, Frequency of clonotypic CD4 cells. C and D, Total intracellular IFN-γ and TNF-α expression, respectively, following in vitro restimulation. B and C, n = 3 for all groups.

FIGURE 3

CY and IL-2 blockade of effector CD4 cell tolerization is transient. A total of 5 × 10⁵ resting effector clonotypic CD4 cells was retransferred into C3-HA^low/high secondary recipients and processed as in Fig. 1, but were analyzed 8 days after retransfer. A, Frequency of clonotypic CD4 cells. B and C, Total intracellular IFN-γ and TNF-α expression, respectively, following in vitro restimulation in the indicated adoptive retransfer recipient groups compared with primary effectors. n = 4 for each group, except for the CY plus IL-2-treated C3-HA^high group in which n = 1 (*, refer to text).

FIGURE 4

CY plus IL-2 treatment enhances self-Ag-induced effector CD4 cell IFN-γ expression in nonlymphoid tissues. A total of 1 × 10⁶ clonotypic effector CD4 cells was retransferred into CY plus IL-2-treated or nontreated C3-HA^high or NT recipients and recovered from lungs following perfusion 4 days later to assess accumulation and in situ IFN-γ expression. A, Representative histograms of IFN-γ expression. B, Total IFN-γ expression in treated (n = 4) and nontreated (n = 3) C3-HA^high recipients. C, Frequency of clonotypic CD4 cells. n = 4 for treated C3-HA^high recipients, n = 3 for nontreated C3-HA^high recipients, n = 2 for treated NT recipients, and n = 3 for nontreated NT recipients. MF, Mean fluorescence.

FIGURE 5

The ability of adoptively retransferred clonotypic effector CD4 cells to induce death in CY plus IL-2-treated self-HA-expressing recipients requires parenchymal Ag presentation. Native C3-HA^high and d → b C3-HA^high bone marrow chimeras treated with CY plus IL-2 received adoptive retransfers of 1.6 × 10⁶ resting effector clonotypic CD4 cells (n = 5 for each group). The percentage of surviving mice in each group over a 9-day period is shown in a Kapler-Meier plot.

FIGURE 6

CY can impede effector CD4 cell tolerization independently of its ability to induce lymphopenia. A total of 5 × 10⁵ resting effector clonotypic CD4 cells was relabeled with CFSE and adoptively retransferred into C3-HA^high secondary adoptive transfer recipients that were either nontreated (−), treated once the day before adoptive retransfer with 180 mg/kg CY, or were treated with CY and also received pooled lymph node and spleen single-cell suspensions containing 2.5 × 10⁷ T cells (CY + T). Four days after retransfer, the clonotypic CD4 cells were recovered from spleens for analysis. A, Frequency of clonotypic CD4 cells. B, Representative histograms showing clonotypic CD4 cell intracellular IFN-γ expression following in vitro restimulation with HA peptide-pulsed APCs, and C, total IFN-γ expression is presented as in Fig. 1. n = 4 for each recipient group. MF, Mean fluorescence.