Jorgen Kjaergaard

Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes.

Recent studies have identified a unique population of CD4+CD25+ regulatory T cells that is crucial for the prevention of spontaneous autoimmune diseases. Further studies demonstrated that depletion of CD4+CD25+ T cells enhances immune responses to nonself antigens. Because immune responses to malignant tumors are weak and ineffective, depletion of regulatory T cells has been reported to result in tumor regression. In the current study, using the weakly immunogenic MCA205 sarcoma and the poorly immunogenic B16/BL6/D5 (D5) melanoma, depletion of CD4+CD25+ T cells by the administration of anti-CD25 monoclonal antibodies (mAb), PC61 induced some tumor growth retardation, but all mice eventually succumbed to tumors. In our laboratory, immunotherapy by the transfer of tumor-immune T cells has demonstrated potent antitumor effects. A reliable source of tumor-reactive T cells has been lymph nodes (LN) draining progressive tumors. Therapeutic effector T cells can be generated by in vitro activation of draining LN cells with anti-CD3 mAb followed by culture in interleukin-2. In this system, PC61 mAb depletion of CD4+CD25+ T cells before or on day 8 of tumor growth resulted in increased sensitization in the draining LN. The therapeutic efficacy of activated tumor-draining LN cells from mAb depleted mice increased approximately three fold while maintaining specificity when tested in adoptive immunotherapy of established pulmonary metastases. Specific interferon-gamma secretion by LN T cells from mice treated with PC61 mAb 1 day before tumor inoculation increased significantly. However, this increase was not demonstrated with LN T cells from mice treated on day 8 despite their enhanced therapeutic reactivities. Our results indicate that although the antitumor immunity enhanced by the depletion of CD4+CD25+ T cells is insufficient to eradicate tumors, it augments the sensitization of immune T cells in the draining LN, thus, facilitating adoptive immunotherapy.

Hypoxia-Adenosinergic Immunosuppression: Tumor Protection by T Regulatory Cells and Cancerous Tissue Hypoxia

Cancerous tissue protection from tumor-recognizing CD8+ and CD4+ T cells (antitumor T cells) limits the therapeutic potential of immunotherapies. We propose that tumor protection is to a large extent due to (a) inhibition of antitumor T cells by hypoxia-driven accumulation of extracellular adenosine in local tumor microenvironment and due to (b) T regulatory cell-produced extracellular adenosine. The adenosine triggers the immunosuppressive signaling via intracellular cyclic AMP–elevating A2A adenosine receptors (A2AR) on antitumor T cells. In addition, the activated antitumor T cells in hypoxic tumor microenvironment could be inhibited by elevated levels of immunosuppressive hypoxia-inducible factor-1α. Complete rejection or tumor growth retardation was observed when A2AR has been genetically eliminated or antagonized with synthetic drug or with natural A2AR antagonist 1,3,7-trimethylxanthine (caffeine). The promising strategy may be in combining the anti-hypoxia-adenosinergic treatment that prevents inhibition of antitumor T cells by tumor-produced and T regulatory cell-produced adenosine with targeting of other negative regulators, such as CTL antigen-4 blockade. Observations of tumor rejection in mice and massive prospective epidemiologic studies support the feasibility of anti-hypoxia-adenosinergic combined immunotherapy.

Immunological mechanisms of the antitumor effects of supplemental oxygenation.

Respiratory hyperoxia stimulates lung tumor regression by promoting T cell infiltration into the tumors and decreasing immunosuppression. Paving the way for intratumoral T cells Tumors often express unusual antigens and are surrounded by immune cells. Unfortunately, this immune surveillance is imperfect and does not always prevent the tumors from growing. In addition, tumors are often hypoxic, because their rapid growth outstrips that of their blood and oxygen supply. Now, Hatfield et al. have linked these two phenomena by demonstrating that T cells avoid going into the hypoxic areas of tumors. The authors have also shown a way to overcome this problem in mice with lung tumors by having the animals breathe supplementary oxygen. Having a higher concentration of oxygen throughout the body improved the oxygenation inside the tumors, allowing immune cells to enter the tumors and attack them, extending the animals’ survival. Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia–A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor–β (TGF-β), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell– and natural killer cell–dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.

Augmentation Versus Inhibition: Effects of Conjunctional OX-40 Receptor Monoclonal Antibody and IL-2 Treatment on Adoptive Immunotherapy of Advanced Tumor

Therapeutic efficacy of adoptive immunotherapy of malignancies is proportional to the number of effector T cells transferred. Traditionally, exogenous IL-2 treatment has been used to promote the survival and function of transferred cells. Recently, we described the therapeutic effects of in vivo ligation of the costimulatory receptor, OX-40R, on activated T cells during early tumor growth. In this study, we examined the effects of IL-2 and OX-40R mAb on adoptive immunotherapy of advanced tumors. For treatment of 10-day 3-methylcholanthrene 205 pulmonary metastases, systemic transfer of 50 × 106 activated tumor-draining lymph node T cells resulted in >99% reduction of metastatic nodules. With either IL-2 or OX-40R mAb conjunctional treatment, only 20 × 106 cells were required. Advanced 10-day 3-methylcholanthrene 205 intracranial tumors could be cured by the transfer of 15 × 106 L-selectinlow T cells derived from draining lymph nodes. In this situation, IL-2 administration inhibited therapeutic effects of the transferred cells. By contrast, 5 × 106 T cells were sufficient to cure all mice if OX-40R mAb was administrated. Studies on trafficking of systemically transferred T cells revealed that IL-2, but not OX-40R mAb, impeded tumor infiltration by T cells. Tumor regression required participation of both CD4 and CD8 T cells. Because only CD4 T cells expressed OX-40R at cell transfer, direct CD4 T cell activation is possible. Alternatively, OX-40R might be up-regulated on transferred T cells at the tumor site, rendering them reactive to the mAb. Our study suggests OX-40R mAb to be a reagent of choice to augment T cell adoptive immunotherapy in clinical trials.

Comparative analysis of antigen loading strategies of dendritic cells for tumor immunotherapy.

Dendritic cells (DCs) loaded with antigens can effectively stimulate host immune responses to syngeneic tumors, but there is considerable controversy as to which forms of antigen-loading are most immunogenic. Here, the authors compared immunotherapeutic reactivities of DCs loaded with a variety of antigen preparations. Because DC maturation stages affect their capacities of antigen processing and presentation, two DC populations were used for the current analysis: in vivo Flt-3 ligand-induced mature DCs and in vitro bone marrow-derived DCs, which were less mature. To facilitate a direct comparison, the LacZ gene-transduced B16 melanoma model system was used, where β-galactosidase served as the surrogate tumor-rejection antigen. DC loading strategies included pulsing with the β-galactosidase protein, H-2Kb restricted peptide, tumor cell lysate, and irradiated tumor cells and fusion of DCs with tumor cells. Our results demonstrated that electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. For the treatment of 3-day established pulmonary tumor nodules, a single intranodal vaccination plus IL-12 resulted in a significant reduction of metastatic nodules, while other DC preparations were only marginally effective. Immunotherapy mediated by the fusion cells was tumor antigen-specific. Consistent with their therapeutic activity, fusion hybrids were the most potent stimulators to induce specific IFN-γ secretion from immune T cells. Furthermore, fusion cells also stimulated a small amount of IL-10 production from immune T cells. However, this IL-10 secretion was also induced by other DC preparations and did not correlate with in vivo therapeutic reactivity.

Tumor-Induced L-Selectinhigh Suppressor T Cells Mediate Potent Effector T Cell Blockade and Cause Failure of Otherwise Curative Adoptive Immunotherapy

Tumor-specific effector T cells (TE) are naturally sensitized within the L-selectinlow (CD62Llow) fraction of tumor-draining lymph nodes (TDLN). Whether isolated from day 9 (D9) or day 12 (D12) TDLN, 5 million L-selectinlow TE could be culture activated and adoptively transferred to achieve complete rejection of established intradermal, pulmonary, and brain tumors. Surprisingly, although 25 million unfractionated T cells from D9 TDLN were equally effective, even 100 million unfractionated T cells from D12 TDLN seldom prevented lethal intradermal tumor progression, despite a pronounced therapeutic excess of TE. This highly reproducible treatment failure was due to cotransfer of tumor-induced, L-selectinhigh suppressor T cells (TS) which were also present in D12 TDLN. In contrast, D9 TDLN and normal spleens lacked L-selectinhigh TS. Only those L-selectinhigh D12 TDLN T cells that down-regulated L-selectin during culture activation were suppressive in vivo and in vitro, and, like L-selectinlow TE, trafficked promptly into tumors following i.v. administration. This is the first demonstration that adoptive immunotherapy can fail as a direct result of passenger TS that share certain phenotypic and trafficking features of TE, even when otherwise curative doses of TE have been administered. Furthermore, in contrast to recently described CD4+CD25+ TS and plasmacytoid dendritic cell-activated TS, tumor-induced L-selectinhigh TS prevent tumor rejection via blockade of sensitized, activated TE rather than via afferent blockade.

Everolimus (RAD) Inhibits In Vivo Growth of Murine Squamous Cell Carcinoma (SCC VII)

Objective: Everolimus (RAD) is an mTOR inhibitor closely related to rapamycin. A potent immunosuppressive agent, it has also shown evidence of antineoplastic properties. SCC VII is a spontaneously arising murine squamous cell carcinoma line. This study examines the effect of everolimus on SCC VII proliferation. The data may provide support for the use of everolimus in transplant recipients with a history of malignancy.

Helper-independent, L-selectin(low) CD8+ T cells with broad anti-tumor efficacy are naturally sensitized during tumor progression

We recently reported that the CD4+ T cell subset with low L-selectin expression (CD62Llow) in tumor-draining lymph nodes (TDLN) can be culture activated and adoptively transferred to eradicate established pulmonary and intracranial tumors in syngeneic mice, even without coadministration of IL-2. We have extended these studies to characterize the small subset of L-selectinlow CD8+ T cells naturally present in TDLN of mice bearing weakly immunogenic tumors. Isolated L-selectinlow CD8+ T cells displayed the functional phenotype of helper-independent T cells, and when adoptively transferred could consistently eradicate, like L-selectinlow CD4+ T cells, both established pulmonary and intracranial tumors without coadministration of exogenous IL-2. Whereas adoptively transferred L-selectinlow CD4+ T cells were more potent on a cell number basis for eradicating 3-day intracranial and s.c. tumors, L-selectinlow CD8+ T cells were more potent against advanced (10-day) pulmonary metastases. Although the presence of CD4+ T cells enhanced generation of L-selectinlow CD8+ effector T cells, the latter could also be obtained from CD4 knockout mice or normal mice in vivo depleted of CD4+ T cells before tumor sensitization. Culture-activated L-selectinlow CD8+ T cells did not lyse relevant tumor targets in vitro, but secreted IFN-γ and GM-CSF when specifically stimulated with relevant tumor preparations. These data indicate that even without specific vaccine maneuvers, progressive tumor growth leads to independent sensitization of both CD4+ and CD8+ anti-tumor T cells in TDLN, phenotypically L-selectinlow at the time of harvest, each of which requires only culture activation to unmask highly potent stand-alone effector function.

Tumor-Dendritic Cell Fusion as a Basis for Cancer Immunotherapy

OBJECTIVE: To establish the basis for use of allogeneic dendritic-tumor fusion cells. STUDY DESIGN: Fusion cells were created by electrofusion. We used 2 allogeneic murine tumor lines (D5 and 4T1) that were virally transduced to express the antigen (β-galactosidase) as a surrogate tumor marker. RESULTS: Cross-immunization was achieved with irradiated allogenic tumor cells. Successful electrofusion of dendritic cells and tumor cells was confirmed by using fluorescence-activated cell sorting and cytospin. Significant responses were shown in immunized mice against tumor challenge and established 3-day pulmonary metastasis with fusion cells. CONCLUSIONS: Allogeneic tumor sharing a common tumor antigen can immunize against syngeneic tumor challenge. Fusion cells showed successful immunization against tumor challenge and showed regression of 3-day established pulmonary metastasis. SIGNIFICANCE: These preclinical studies provide evidence that an allogenic tumor-dendritic cell fusion vaccine is a valid approach for head and neck cancer immunotherapy. (Otolaryngol Head Neck Surg 2005;132:755-64.)

Memory T Cells Originate from Adoptively Transferred Effectors and Reconstituting Host Cells after Sequential Lymphodepletion and Adoptive Immunotherapy

Adoptive transfer of tumor-specific effector T cells induces regression of advanced tumors and induces a long term memory response; however, the origin of this response has not been clearly defined. In this study Thy1.2+ mice bearing advanced MCA-205 tumors were treated with sublethal total body irradiation, followed by adoptive transfer of congenic Thy1.1+ T cells that had been sensitized to tumor in vivo and then activated ex vivo with anti-CD3, IL-2, and IL-7. Splenocytes were recovered >140 days after the initial therapy, and the L-selectinlow memory cell subset was separated into host Thy1.2+ and transferred Thy1.1+ cells and restimulated ex vivo. Both adoptively transferred Thy1.1+ cells as well as reconstituted host Thy1.2+ cells could specifically eliminate MCA-205 pulmonary metastases. Interestingly, hosts with partial responses followed by tumor recurrence nevertheless harbored memory cells that could be isolated and numerically amplified ex vivo to regenerate potent effector function. Memory cells were recovered after adoptive transfer into lymphodepleted nontumor-bearing hosts, indicating that they were not dependent on continued Ag exposure. These experiments establish that rapid ex vivo expansion of tumor Ag-primed T cells does not abrogate their capacity to become long-lived memory cells. Moreover, immune-mediated tumor regression coincident with lymphoid reconstitution produces another wave of host memory cells. These data suggest an approach to rescuing antitumor immune function even in hosts with long-standing progressive tumor through restorative ex vivo activation.