Expanding roles for CD4 T cells and their subpopulations in tumor immunity and therapy.

The importance of CD4 T cells in orchestrating the immune system and their role in inducing effective T cell-mediated therapies for the treatment of patients with select established malignancies are undisputable. Through a complex and balanced array of direct and indirect mechanisms of cellular activation and regulation, this functionally diverse family of lymphocytes can potentially promote tumor eradication, long-term tumor immunity, and aid in establishing and/or rebalancing immune cell homeostasis through interaction with other immune cell populations within the highly dynamic tumor environment. However, recent studies have uncovered additional functions and roles for CD4 T cells, some of which are independent of other lymphocytes, that can not only influence and contribute to tumor immunity but paradoxically promote tumor growth and progression. Here, we review the recent advances in our understanding of the various CD4 T cell lineages and their signature cytokines in disease progression and/or regression. We discuss their direct and indirect mechanistic interplay among themselves and with other responding cells of the antitumor response, their potential roles and abilities for "plasticity" and memory cell generation within the hostile tumor environment, and their potentials in cancer treatment and immunotherapy.

Cytotoxic T-lymphocyte immunotherapy for ovarian cancer: a pilot study.

The objective was to evaluate the toxicity and feasibility of intraperitoneal infusion of tumor-specific cytotoxic T lymphocytes (CTL) as therapy for recurrent ovarian cancer, and to determine if repetitive cycles of CTL generation and infusion measurably increases the host's ovarian cancer immune response. In this study, 7 subjects with recurrent ovarian cancer confined to the peritoneal cavity underwent up to 4 cycles, each cycle beginning with a leukapheresis for collection of precursor lymphocytes, which were stimulated in vitro with mucin 1, a tumor-specific antigen found commonly in ovarian cancer cells. The resulting new CTL for each cycle were reintroduced into the host by intraperitoneal infusion. Immunologic parameters (killer cells, cytokine production, memory T lymphocytes, and natural killer cells) were studied. Toxicity, CA-125, and survival data were also evaluated. The tumor marker CA-125 was nonstatistically significantly reduced after the first month of immunotherapy. However, after that it rose. Killer cells, cytokine production, and memory T lymphocytes increased after the first cycle of stimulation, but plateaued or reduced thereafter. The percent of natural killer cells inversely correlated with other immune parameters. Median survival was 11.5 months. One subject is free of disease since December, 2000. Multiple cycles, beyond 1 cycle, of T-cell stimulation followed by adoptive T-cell infusion, may not enhance the in vivo immune response.

Immunotherapy with IL-10- and IFN-γ-producing CD4 effector cells modulate "Natural" and "Inducible" CD4 TReg cell subpopulation levels: observations in four cases of patients with ovarian cancer.

Adoptive T cell therapy for cancer patients optimally requires participation of CD4 T cells. In this phase I/II study, we assessed the therapeutic effects of adoptively transferred IL-10- and IFN-γ-producing CD4 effector cells in patients with recurrent ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 effector cell generation, we show that three monthly treatment cycles of autologous T cell restimulation and local intraperitoneal re-infusion-modulated T cell-mediated immune responses that were associated with enhanced patient survival. One patient remains disease-free, another patient experienced prolonged survival for nearly 16 months with recurrent disease, and two patients expired within 3-5 months following final infusion. Prolonged survivors showed elevated levels of systemic CD3(+)CD4(+)CD25(+) and CD3(+)CD4(+)CD25(-) T cells when compared to that of pre-treatment levels and similarly treated short-term survivors. Such cell populations among these patients contained variable levels of "Inducible" Tr1 (CD4(+)CD25(-)FoxP3(-)IL-10(+)) and "Natural" (CD4(+)CD25(+)CD45RO(+)FoxP3(+)) TReg cell numbers and ratios that were associated with prolonged and/or disease-free survival. Moreover, peptide-restimulated T cells from these patients showed an elevation in both IFN-γ production, memory cell phenotype, and select TNF family ligands associated with enhanced T cell survival and apoptosis-inducing activities. This suggests that intraperitoneally administered Th1-like cells, producing elevated levels of IL-10, may require and/or induce differential levels of distinct systemic TReg subpopulations that influence, in part, long-term tumor immunity and enhanced memory/effector CD4-mediated therapeutic potentials. Furthermore, treatment efficacy and enhanced memory cell phenotype did not appear to be dependent on TReg cell numbers but upon ratios of "Inducible" and "Natural" TReg subpopulations.

Autologous MUC1-specific Th1 effector cell immunotherapy induces differential levels of systemic TReg cell subpopulations that result in increased ovarian cancer patient survival.

Adoptive T cell immunotherapy using autologous lymphocytes is a viable treatment for patients with cancer and requires participation of Ag-specific CD4 and CD8 T cells. Here, we assessed the immunotherapeutic effects of autologous MUC1 peptide-stimulated CD4(+) effector cells following adoptive transfer in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4(+)/Th1 effector cell generation, we show that three monthly treatment cycles of peripheral blood T cell restimulation and intraperitoneal re-infusion selectively modulated endogenous T cell-mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease-free, another patient survived long-term for nearly 16 months with recurrent disease and two patients expired within 3-5 months following final infusion. Although PBL from all patients showed elevated MUC1 cytolytic activity following therapy, such responses did not correlate with therapeutic efficacy. Long-term survivors showed elevated levels of systemic memory (CD45RO) and naïve (CD45RA) CD3/CD4/CD25(+) T cells when compared to that of pre-treatment levels and similarly treated short-term survivors. Such cells co-expressed different levels of Foxp3 and CTLA-4 that resulted in progressively lower systemic Foxp3/CTLA-4 memory T cell ratios that further correlated with disease-free survival. Lastly, these patients showed elevated levels of MUC1-specific T cells expressing the CCR5 and CCR1 chemokine receptors and the chemokine CCL4 associated with Th1 cell differentiation/memory. We suggest that effective immunotherapy with autologous MUC1-stimulated CD4(+) effector cells induces differential levels of systemic "Ag-experienced" and "Ag-inexperienced" CD4/CD25(+) TReg cell subpopulations that influence long-term tumor immunity in ovarian cancer patients.

Ag-specific type 1 CD8 effector cells enhance methotrexate-mediated antitumor responses by modulating endogenous CD49b-expressing CD4 and CD8 T effector cell subpopulations producing IL-10.

The chemotherapeutic agent methotrexate is widely used in the treatment of breast cancer. Although its mechanism-of-action has been defined, less is known about its interaction with Ag-specific T cell-mediated antitumor responses. Type 1 CD8 T cell-mediated immune responses (Tc1) are cytolytic, produce IFN-gamma and are associated with effective antitumor responses. Using a murine transgenic TCR tumor model, we show that single-dose-treatment with methotrexate enhanced CD8-mediated type 1 antitumor responses when administered three days prior to Tc1 effector cell transfer. Co-treatment with methotrexate not only enhanced donor Tc1 cell accumulation and persistence at sites of primary tumor growth, but also promoted elevated levels of activated CD25(+) expressing donor TIL cells. This correlated with a marked decrease in the appearance of endogenous differentiated (CD44(High)) CD3/CD8/CD49b and CD3/CD4/CD49b tumor-infiltrating effector T cells at both early (Days 1-8) and late (Days 12-20) stages following treatment when compared to that of corresponding groups receiving either MTX or Tc1 cell transfer alone. Moreover, such cellular response kinetics appeared to further correlate with the down-regulation of endogenous CD4/CD44(High)/CD49b effector T cells producing IL-10 and delays in tumor growth in vivo. This suggested that Ag-specific Tc1 cell transfer, in combination with chemotherapy, can enhance antitumor responses by modulating select CD49b-expressing T effector/memory cell subpopulations involved in homeostasis and immune tolerance within the tumor environment. These studies offer insight into mechanisms that enhance T cell-based immunotherapy in cancer. Supplementary materials are available for this article. Go to the publisher's online edition of Immunological Investigations for the following free supplemental resource(s): Addendum 1.

Ag-specific type 1 CD8 effector cells enhance methotrexate-mediated antitumor responses by modulating differentiated T cell localization, activation and chemokine production in established breast cancer.

The chemotherapeutic agent methotrexate is widely used in the treatment of breast cancer. Although its mechanism-of-action has been defined, less is known about its interaction with T cell-mediated antitumor responses. Type 1 CD8 T cell-mediated immune responses (Tc1) are cytolytic, produce IFN-gamma and are associated with effective antitumor responses. Using a murine transgenic TCR tumor model, we show that single-dose treatment with methotrexate enhanced CD8-mediated type 1 antitumor responses when administered 3 days prior to Tc1 effector cell transfer. Co-treatment with methotrexate not only enhanced donor Tc1 cell accumulation and persistence at sites of primary tumor growth, but also promoted elevated levels of activated donor TIL cells. This markedly enhanced the appearance of endogenous differentiated (CD44(High)) CD8 tumor-infiltrating cells when compared to that of corresponding groups receiving either MTX or Tc1 cell transfer alone. Such cells were acutely activated as defined by co-expression of surface markers associated with TCR engagement (CD69) and T cell activation (CD25) at both early (days 1-8) and late (days 12-20) stages following treatment. Conversely, such animals showed an early decrease in CD4(+)/CD44(High)/CD25(+)/CD69(+) T cells that correlated with delays in tumor growth in vivo. Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IP-10 in vivo. This suggested that Tc1 cell transfer, in combination with chemotherapy, can enhance antitumor responses by modulating immunoregulatory T cells involved in homeostasis and immune tolerance within the tumor environment. These studies offer insight into mechanisms that enhance T cell-based immunotherapy in cancer.

CD8-mediated type 1 antitumor responses selectively modulate endogenous differentiated and nondifferentiated T cell localization, activation, and function in progressive breast cancer.

CD8 T cell-mediated immune responses fall into two distinct types based on effector cell-derived cytokine production. Type I CD8 T cells (Tc1) produce IFN-gamma, whereas type 2 cells (Tc2) secrete IL-4, IL-5, IL-10, and GM-CSF. Using a murine TCR transgenic T cell/breast tumor model, we show that adoptively transferred Ag-specific Tc1 cells are more effective in delaying mammary tumor growth and progression than that of functionally distinct Tc2 cells. Donor Tc1 cells administered 7 days posttumor challenge localized and persisted at sites of primary tumor growth with antitumor responses that were dependent, in part, on effector cell-derived IFN-gamma. Tc1-mediated responses markedly enhanced the appearance and local accumulation of highly differentiated (CD44(high)) CD4 and CD8 endogenous tumor-infiltrating T cells when compared with that of untreated tumor-bearing mice. Conversely, Tc1 cell transfer markedly delayed the appearance of corresponding nondifferentiated (CD44(low)) endogenous T cells. Such cells were acutely activated as defined by coexpression of surface markers associated with TCR engagement (CD69) and early T cell activation (CD25). Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IFN-gamma-inducible protein-10 in vivo. This suggested that CD8-mediated type 1 antitumor responses cannot only promote accumulation of distinct endogenous CD4 and CD8 T cell subpopulations, but also facilitate and preferentially modulate their localization kinetics, persistence, states of activation/differentiation, and function within the primary tumor environment at various stages of tumor progression. These studies offer insight into potential mechanisms for enhancing T cell-based immunotherapy in breast cancer.

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer.

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.

Tc1 and Tc2 effector cell therapy elicit long-term tumor immunity by contrasting mechanisms that result in complementary endogenous type 1 antitumor responses.

Cytolytic CD8(+) effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8(+) T cells (Tc1) secrete IFN-gamma, whereas type 2 CD8(+) T cells (Tc2) secrete IL-4 and IL-5. Both effector cell subpopulations display predominantly perforin-dependent cytolysis in vitro. Using an OVA-transfected B16 lung metastases model, we show that adoptively transferred OVA-specific Tc1 and Tc2 cells induce considerable suppression, but not cure, of pulmonary metastases. However, long-term tumor immunity prolonged survival times indefinitely and was evident by resistance to lethal tumor rechallenge. At early stages after therapy, protection by Tc2 and Tc1 effector cells were dependent in part on effector cell-derived IL-4, IL-5, and IFN-gamma, respectively. Whereas effector cell-derived perforin was not necessary. Over time the numbers of both donor cells diminished to low, yet still detectable, levels. Concomitantly, Tc1 and Tc2 effector cell therapies potentiated endogenous recipient-derived antitumor responses by inducing 1) local T cell-derived chemokines associated with type 1-like immune responses; 2) elevated levels of recipient-derived OVA tetramer-positive CD8 memory T cells that were CD44(high), CD122(+), and Ly6C(high) that predominantly produced IFN-gamma and TNF-alpha; and 3) heightened numbers of activated recipient-derived Th1 and Tc1 T cell subpopulations expressing CD25(+), CD69(+), and CD95(+) cell surface activation markers. Moreover, both Tc2 and Tc1 effector cell therapies were dependent in part on recipient-derived IFN-gamma and TNF-alpha for long-term survival and protection. Collectively, Tc1 and Tc2 effector cell immunotherapy mediate long-term tumor immunity by different mechanisms that subsequently potentiate endogenous recipient-derived type 1 antitumor responses.

Effector cell-derived lymphotoxin alpha and Fas ligand, but not perforin, promote Tc1 and Tc2 effector cell-mediated tumor therapy in established pulmonary metastases.

Cytolytic CD8(+) effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8(+) T cells (Tc1) secrete IFN-gamma, whereas type 2 CD8(+) T cells (Tc2) secrete interleukin (IL)-4 and IL-5. Although both effector cell subpopulations display Fas ligand (FasL) and tumor necrosis factor (TNF), tumor lysis is predominantly perforin dependent in vitro. Using an ovalbumin-transfected B16 lung metastasis model, we show that heightened numbers of adoptively transferred ovalbumin-specific Tc1 and Tc2 cells accumulated at the tumor site by day 2 after therapy and induced tumor regression that enhanced survival in mice with pulmonary metastases. Transfer of either TNF-alpha- or perforin-deficient Tc1 or Tc2 effector cells generated from specified gene-deficient mice showed no differences in therapeutic efficiency when compared with corresponding wild-type cells. In contrast, both Tc1 and Tc2 cells, derived from either FasL or TNF-alpha/lymphotoxin (LT) alpha double knockout mice, showed that therapeutic effects were dependent, in part, on effector cell-derived FasL or LTalpha. Six days after effector cell therapy, elevated levels of activated endogenous CD8/CD44(High) and CD4/CD44(High) T cells localized and persisted at sites of tumor growth, whereas donor cell numbers concomitantly decreased. Both Tc1 and Tc2 effector cell subpopulations induced endogenous antitumor responses that were dependent, in part, on recipient-derived IFN-gamma and TNF-alpha. However, neither effector cell-mediated therapy was dependent on recipient-derived perforin, IL-4, IL-5, or nitric oxide. Collectively, tumor antigen-specific Tc1 and Tc2 effector cell-mediated therapy is initially dependent, in part, on effector cell-derived FasL or LTalpha that may subsequently potentiate endogenous recipient-derived type 1 antitumor responses dependent on TNF-alpha and IFN-gamma.