OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection.

Regulatory T (T reg) cells are the major obstacle to cancer immunotherapy, and their depletion promptly induces conversion of peripheral precursors into T reg cells. We show that T reg cells can be functionally inactivated by OX40 triggering. In tumors, the vast majority of CD4(+) T cells are Foxp3(+) and OX40(bright). However, intratumor injection of the agonist anti-OX40 monoclonal antibody (mAb) OX86, but not anti-CD25 mAb, induces tumor rejection in 80% of mice, an effect that is abrogated by CD8 depletion. Upon intratumor OX40 triggering, increased numbers of infiltrating dendritic cells (DCs) migrate to draining lymph nodes and generate a new wave of tumor-specific cytotoxic T lymphocytes, as detected by tetramer and CD44 staining of node CD8(+) T lymphocytes. Tumor-bearing Rag1-knockout (KO) mice reconstituted with OX40-deficient T reg cells and wild-type (WT) effector T cells, or the reciprocal combination, showed that both T reg and effector T cells must be triggered via OX40 for the tumor to be rejected. Accordingly, WT but not OX40-KO mice receiving intratumor coinjection of OX86 and ovalbumin protein were able to revert tumor-induced tolerization of adoptively transferred OX40-competent OTII T lymphocytes. In conclusion, OX40-mediated inactivation of T reg cell function unleashes nearby DCs, allowing them to induce an adaptive immune response. In addition, the known OX40-dependent delivery of fitness signals to activated T cells is boosted by concurrent T reg cell inhibition. OX40 triggering thus has multiple effects that converge to mediate tumor rejection.

Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells.

Indoleamine 2,3-dioxygenase (IDO) is a novel immunosuppressive agent expressed in some subsets of normal and neoplastic cells, including acute myeloid leukemia (AML) cells. Here, we show that IDO expression correlates with increased circulating CD4+CD25+FOXP3+ T cells in patients with AML at diagnosis. In vitro, IDO+ AML cells increase the number of CD4+ CD25+ T cells expressing surface CTLA-4 and FOXP3 mRNA, and this effect is completely abrogated by the IDO inhibitor, 1-methyl tryptophan (1-MT). Purified CD4+CD25+ T cells obtained from coculture with IDO+ AML cells act as T regulatory (T(reg)) cells because they do not proliferate, do not produce interleukin (IL)-2, and inhibit naive T-cell proliferation. Coculture with IDO+AML cells results in the conversion of CD4+CD25- into CD4+CD25+ T cells, which is completely abrogated by 1-MT. Moreover, in mice, intrasplenic injection of IDO+ leukemia/ lymphoma A20 cells induces the expansion of bona fide T(reg) cells by conversion of CD4+CD25- T cells; this effect is counteracted by 1-MT treatment. These data indicate that AML cells induce T-cell tolerance by directly converting CD4+CD25- T cells into CD4+CD25+ T(reg) cells through an IDO-dependent mechanism.

Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent.

The CD25- and CD25+ CD4 T-lymphocyte compartments are tightly regulated. We show here that tumors break such balance, increasing the number of CD4+CD25+ T cells in draining lymph node and spleen but not contralateral node of tumor-bearing mice. Tumor injection in thymectomized and CD25-depleted mice shows that CD4+CD25+ T-cell expansion occurs even in the absence of the thymus and independently from proliferation of preexisting CD25+ T cells. These newly generated cells are bona fide regulatory T cells (T reg) in terms of Foxp3 expression and suppression of CD3-stimulated or allogeneic effector cell proliferation. Transfer of congenic Thy1.1 CD4+CD25- T cells, from mice treated or not with vinblastine, into tumor-bearing or tumor-free mice and analysis of recovered donor lymphocytes indicate that conversion is the main mechanism for acquiring the expression of CD25 and Foxp3 through a process that does not require proliferation. Although conversion of CD4+CD25- T cells for generation of T regs has been described as a natural process that maintains peripheral T-reg population, this process is used by the tumor for immune escape. The prompt recovery of T regs from monoclonal antibody-mediated CD25 depletion in tumor-bearing mice suggests attempts able to inactivate rather than deplete them when treating existing tumors.

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine.

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

CD40/CD40L interaction regulates CD4+CD25+ T reg homeostasis through dendritic cell-produced IL-2.

CD4+CD25+ regulatory T cells (T reg) development and homeostasis require IL-2 and costimulation through same TNF-receptor family members. CD40KO mice have reduced number of T reg in peripheral blood, thymus and spleen. Herein we show that naive T reg express low basal level of CD40L that is upregulated upon TCR-triggered mediated activation. Treatment of wt mice with Ab blocking CD40/CD40L interaction results in a fast decrease in T reg number that rapidly recovers upon Ab withdrawal. CFSE-labeled T reg from wt mice injected into CD40KO, but not wild-type (wt) mice, showed reduced survival and proliferation in homeostatic setting. In vitro, dendritic cells from CD40KO mice but not wt mice produce diminished amount of IL-2 upon T reg encounter and are impaired in expanding T reg, a defect corrected by the addition of rIL-2. Accordingly, four daily IL-2 administrations to CD40KO mice normalize T reg number by promoting both their survival and homeostatic proliferation. Such IL-2 effect is transient since T reg number returns to the low constitutive level described in CD40KO mice within 5 days upon IL-2 withdrawal thus suggesting that IL-2 is persistently needed to assure T reg homeostasis.

Triggering of OX40 (CD134) on CD4(+)CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR.

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor family that is transiently expressed on T cells after T-cell receptor (TCR) ligation. Both naive and activated CD4(+)CD25+ regulatory T cells (T reg's) express OX40 but its functional role has not been determined. Since glucocorticoid-induced tumor necrosis factor receptor (GITR), a related TNF receptor family member, influences T reg function, we tested whether OX40 might have similar effect. Triggering either GITR or OX40 on T reg's using agonist antibodies inhibited their capacity to suppress and restored effector T-cell proliferation, interleukin-2 (IL-2) gene transcription and cytokine production. OX40 abrogation of T reg suppression was confirmed in vivo in a model of graft-versus-host disease (GVHD). In a fully allogeneic C57BL/6>BALB/c bone marrow transplantation, GVHD was lethal unless T reg's were cotransferred with the bone marrow and effector T cells. Strikingly, T reg suppression of GVHD was abrogated either by intraperitoneal injection of anti-OX40 or anti-GITR monoclonal antibodies (mAbs) immediately after transfer, or by in vitro pretreatment of T reg's with the same mAbs before transfer. Cumulatively, the results suggest that in addition to controlling memory T-cell numbers, OX40 directly controls T reg-mediated suppression.

Toll-like receptor 4 is not required for the full maturation of dendritic cells or for the degradation of Gram-negative bacteria.

Toll-like receptor 4 (TLR4) has been recently associated with cellular responses to lipopolysaccharide (LPS), and mice mutated in tlr4, such as C57BL/10ScCr or C3H/HeJ mice, become hyporesponsive to LPS. In this study, we have analyzed the capacity of bone marrow-derived dendritic cells (BMDC) from C57BL/10ScCr (ScCr-BMDC) or C3H/HeJ (HeJ-BMDC) mice to respond to LPS or to Gram-negative bacteria. We show that ScCr- or HeJ-BMDC are insensitive to LPS, but can mature in response to live and killed Gram-negative bacteria. Interestingly, only ScCr-BMDC but not HeJ-BMDC, stimulated with bacteria, have reduced capacity to produce pro- and anti-inflammatory cytokines as compared to BMDC from control mice, probably due to genetic defects unrelated to the tlr4 mutation. Nevertheless, ScCr-BMDC and ScCr BM-macrophages (BM-Mphi) phagocytose Salmonella typhimurium similarly to control cells, indicating that TLR4 is not compulsory for bacterial uptake. Moreover, BM-Mphi, but not BM-DC from B10ScCr or C3H/HeJ mice, are impaired in their capacity to kill intracellular bacteria and to produce NO as compared to wild type controls. However, the bacteria killing property of BM-Mphi is completely restored by pretreating the cells with IFN-gamma. Hence, TLR4 plays different roles in DC versus Mphi.