Gregory E. Plautz

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.

T Cell-Mediated Tumor Rejection Displays Diverse Dependence Upon Perforin and IFN-γ Mechanisms That Cannot Be Predicted From In Vitro T Cell Characteristics

Experimental pulmonary metastases have been successfully treated by adoptive transfer of tumor-sensitized T cells from perforin knockout (KO) or Fas/APO-1 ligandKO mice, suggesting a prominent role for secretion of cytokines such as IFN-γ. In the present study we confirmed that rejection of established methylcholanthrene-205 (MCA-205) pulmonary metastases displayed a requirement for T cell IFN-γ expression. However, this requirement could be obviated by transferring larger numbers of tumor-sensitized IFN-γ KO T cells or by immunosensitizing sublethal irradiation (500 rad) of the host before adoptive therapy. Extrapulmonary tumors (MCA-205 s.c. and intracranial) that required adjunct sublethal irradiation for treatment efficacy also displayed no requirement for host or T cell expression of IFN-γ. Nonetheless, rejection of MCA-205 s.c. tumors and i.p. EL-4 tumors, but not MCA-205 pulmonary or intracranial tumors, displayed a significant requirement for T cell perforin expression (i.e., CTL participation). The capacity of T cells to lyse tumor targets and secrete IFN-γ in vitro before adoptive transfer was nonpredictive of the roles of these activities in subsequent tumor rejection. Adoptive therapy studies employing KO mice are therefore indispensable for revealing a diversity of tumor rejection mechanisms that may lack in vitro correlation due to delays in their induction. Seemingly contradictory KO data from different studies are reconciled by the capacity of anti-tumor T cells to rely on alternative mechanisms when treated in larger numbers, the variable participation of CTL at different anatomic locations of tumor, and the apparent capacity of sublethal irradiation to provide a therapeutic alternative to host or T cell IFN-γ production.

T-cell adoptive immunotherapy of metastatic renal cell carcinoma

OBJECTIVES To determine the feasibility and toxicity of the adoptive transfer of ex vivo-activated T lymphocytes that have been sensitized to autologous tumor vaccine in vivo. METHODS Twenty patients with extensive metastatic renal cell carcinoma received systemic adoptive immunotherapy with autologous T cells in the absence of conjunctional interleukin-2 (IL-2) administration. Patients were vaccinated intradermally with irradiated autologous tumor cells and granulocyte-macrophage colony-stimulating factor as an adjuvant to stimulate an immune response. Inguinal lymph nodes draining the vaccine site were surgically removed, and the cells were stimulated with staphylococcal enterotoxin A followed by expansion in 60 IU/mL IL-2, and in some cases additionally stimulated with anti-CD3 monoclonal antibody and IL-2, to obtain a large number of cells. RESULTS The staphylococcal enterotoxin A/IL-2 activation induced vigorous proliferation of T cells (median expansion 26-fold) that were a mixture of CD4 and CD8 T lymphocytes. Activated cells were infused intravenously at doses ranging from 2x10(9) to 9.5x10(10). There was minimal toxicity consisting of grade 1 or 2 fever and nausea, and the entire treatment was delivered as outpatient therapy. One patient had a partial response, one had a mixed response, and 8 had stable disease lasting at least 5 months. CONCLUSIONS Adoptive transfer of ex vivo-activated, tumor vaccine-primed lymph node cells is feasible and is associated with minimal toxicity when used alone. These results warrant further study in a Phase II trial.

Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells

T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vβ families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.

Effects of surgery, general anesthesia, and perioperative epidural analgesia on the immune function of patients with non-small cell lung cancer ☆

STUDY OBJECTIVE To assess preoperative and postoperative immune function in patients undergoing surgical resection of non-small cell lung cancer during general anesthesia and postoperative epidural analgesia. DESIGN Observational single-center study. SETTING University-affiliated academic center. PATIENTS 24 adult, ASA physical status 3 and 4 patients with stage 1, 2, or 3 non-small cell lung cancer. No study patient received preoperative chemotherapy or radiation. INTERVENTIONS Patients underwent thoracotomy with general anesthesia and postoperative epidural analgesia. MEASUREMENTS Bispectral index monitoring, sevoflurane requirements, and intraoperative transfusions were recorded. Total fentanyl consumption and pain (verbal numeric rating scale) were recorded 24 hours after surgery. Preoperative and 24-hour postoperative natural killer cell percentage and function and percentages of natural killer T cells, T helper cells (CD4+), and cytotoxic T lymphocytes (CD8+) were measured. Plasma concentrations of the TH1 cytokine interleukin-2 and interferon-gamma and the TH2 cytokines interleukin-4 were measured at the same time points. RESULTS The percentage (preoperative, 13.07 ± 9.81% vs postoperative, 9.6 ± 6.57%, P < 0.001) and function (preoperative, 31.61 ± 21.96%; postoperative, 13.61 ± 9.36%; P < 0.001) of natural killer cells was significantly decreased after surgery, but the percentage of natural killer T cells, T helper cells (CD4+), and cytotoxic T lymphocytes (CD8+) remained unchanged postoperatively; thus, the CD4/CD8 ratio remained unchanged. Postoperative plasma concentrations of the three cytokines were similar to preoperative levels; therefore, the TH1/TH2 ratio also remained unchanged. CONCLUSIONS Innate immunity is depressed in patients with non-small cell lung cancer after surgical resection, and immunity is not preserved by the use of postoperative epidural analgesia.

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.

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

Abstract The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56lck and p59fyn and proteins of the NF-κB family were analyzed in tumor-draining LN T cells. The levels of p56lck and p59fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56lck and p59fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56lck, p59fyn, and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear κB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-κB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function.

Chylopericardium and chylothorax: Unusual mechanical complications of central venous catheters

Obstruction and thrombosis of major systemic veins can occur due to indwelling central venous catheters. If obstruction of the innominate vein or superior vena cava occurs, lymphatic drainage can be impaired due to an increase in pressure in the thoracic duct and lymphatics. We describe a case where superior vena cava syndrome, chylopericardium and chylothorax occurred in a 16‐year‐old girl due to an indwelling central venous catheter. This was successfully treated with removal of the line, anticoagulation and angioplasty of the innominate vein and superior vena cava.

Potent Effector Function of Tumor-Sensitized L-Selectinlow T Cells against Subcutaneous Tumors Requires LFA-1 co-Stimulation

OBJECTIVE: Animal tumor models have demonstrated that adoptive transfer of tumor-draining lymph node (TDLN) T lymphocytes can cure established tumors in many anatomic sites. However, subcutaneous tumors are relatively refractory and have required maximally tolerated doses of cells. The goals of this study were to determine whether a subset of TDLN T lymphocytes varying in expression of the cell adhesion molecule L-selectin (CD62L) had augmented therapeutic efficacy and to determine the co-stimulatory requirements for trafficking and anti-tumor effector function. STUDY DESIGN: TDLNs were recovered from mice bearing progressive MCA 205 fibrosarcomas, and the T lymphocytes were segregated into CD62Llow and CD62Lhigh subsets and activated ex vivo with anti-CD3 mAb and IL-2. Mice bearing established subcutaneous MCA 205 tumors were treated with activated T cell subsets and in some experiments with additional mAb against cell adhesion molecules. RESULTS: Adoptive transfer of as few as 5 × 10 6 activated cells cured mice bearing 3-day subcutaneous MCA 205 tumors initiated with 6 × 10 6 cells, and the tumors demonstrated a dense infiltrate of CD62Llow cells. In marked contrast, adoptive transfer of 10 times as many T cells derived from the reciprocal CD62Lhigh compartment had no effect on tumor growth. The effector function of the CD62Llow T cells was clearly dependent on co-stimulation through the cell adhesion molecule LFA-1, because anti-LFA-1 mAb completely abrogated the antitumor reactivity of the transferred cells against subcutaneous tumors and inhibited tumor infiltration. In contrast, blockade of ICAM-1, VLA-4, or VCAM-1 had no inhibitory effect on the anti-tumor function. CONCLUSION: These studies demonstrate the high therapeutic activity of the CD62Llow subset of tumor-draining LN T cells against subcutaneous tumors, a relatively refractory site, and confirm the essential role of LFA-1 for effector T cell function. SIGNIFICANCE: Identification of the phenotype and requirements for effector function of T lymphocytes sensitized to tumor antigens has implications for clinical trials of adoptive immunotherapy for head and neck carcinoma using a similar approach.

T cells sensitized with breast tumor progenitor cell vaccine have therapeutic activity against spontaneous HER2/neu tumors

Cancer progenitor cells are critical for tumor initiation and recurrence so they are an important therapeutic target. We tested whether T cells could recognize tumor antigens expressed by breast cancer progenitor cells and acquire therapeutic activity against established metastases or delay onset of spontaneous tumors. Breast tumors were derived from HER2/neu transgenic mice and propagated in vitro under conditions that selected progenitor cells which were then used as an irradiated whole cell vaccine. A minor subset of recently sensitized T cells was isolated from vaccine-draining lymph nodes then activated in vitro to achieve numerical expansion. We show that the tumor progenitor cell vaccines reversed tolerance to a known HER2/neu epitope, otherwise inhibited by Treg cells. Additional shared tumor antigens were recognized because a Neuneg subclone also induced a Th1 type immune response against breast tumors. Adoptive transfer of in vitro activated lymph node T cells-mediated regression of established metastases from multiple independently derived breast tumor lines. Moreover, adoptive transfer of effector T cells into Neu-tolerant mice, months before the onset of spontaneous tumors, significantly postponed tumor development. Interestingly, T-cell-mediated lysis of metastases stimulated an IgG response to HER2/neu as well as other shared antigens. In summary, tumor progenitor cells contain shared antigens which can lead to a cross-protective T-cell response. Moreover, antigens acquired during immune-mediated tumor destruction are presented in a manner conducive to reversal of tolerance and Ig class switching. These complementary effector mechanisms might augment therapy by eliminating refractory breast cancer stem cells.