Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3-genetically modified lymphocytes.

Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti-vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine-specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE-A3(+) melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE-A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV-TK). HSV-TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti-TK and anti-MAGE-A3 T-cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE-A3-specific immune responses showed a clinical benefit. Additionally, we report that responder and non-responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses.

Secondary Lymphedema: Clinical Interdisciplinary Tricks to Overcome an Intriguing Disease.

Secondary lymphedema is a complex pathology which is very impairing to the patient, consisting of fluid accumulation in the tissue, accompanied by alteration of the interstitial fibrous tissue matrix, deposition of cellular debris and local inflammation. It develops mostly in limbs and/or external genitals because of demolishing oncological surgery with excision of local lymph nodes, or it may depend upon inflammatory or infective diseases, trauma, or congenital vascular malformation. Its treatment foresees various approaches, from simple postural attitude to physical therapy, to minimally invasive lymphatic microsurgery. This review focuses on the different types of evolving peripheral lymphedema and describes potential solutions to single objective symptoms. Particular attention is paid to the newest lymphatic microsurgical approaches, such as lymphatic grafting and lympho-venous shunt application, to successfully heal, in the long term, serious cases of secondary lymphedema of limbs or external genitals. The presented data also emphasize the potential role of minimally invasive microsurgery in enhancing the development of newly formed lymphatic meshes, focusing on the need for further accurate research in the development of microsurgical approaches to the lymphatic vascular system.

Peripheral blood lymphocytes genetically modified to express the self/tumor antigen MAGE-A3 induce antitumor immune responses in cancer patients.

Dendritic cell (DC) targeting in vivo has recently been shown to confer strong and protective cytotoxic T lymphocyte (CTL)-based immunity in tumor murine models. Our group has recently demonstrated in preclinical models that the infusion of genetically modified lymphocytes (GMLs) expressing the self/tumor antigen TRP-2 is able to elicit functional TRP-2-specific effectors with antitumor activity by targeting DCs in vivo. Here we have analyzed vaccine- and tumor-specific immune responses of 10 melanoma patients treated with autologous GMLs expressing the cancer germline gene MAGE-A3. Three of 10 patients treated with MAGE-A3-GML showed an increase of circulating anti-MAGE-A3 T cells, and developed skin delayed-type hypersensitivity to MAGE-A3. Interestingly, in 2 of these patients, with progressive and measurable tumors at study entry, anti-MAGE-A3 T cells were detected not only in the blood but also within tumors resected after vaccination. These results demonstrate that the infusion of MAGE-A3-GML elicits antitumor T cells, which are capable of trafficking to inflamed tissues and of infiltrating tumors. Clinical studies on a larger group of patients are needed to evaluate the clinical efficacy of such a strategy.

Lymphocytes genetically modified to express tumor antigens target DCs in vivo and induce antitumor immunity.

The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.

Investigation on the role of biallelic variants in VEGF-C found in a patient affected by Milroy-like lymphedema.

BACKGROUND: Milroy-like disease is the diagnostic definition used for patients with phenotypes that resemble classic Milroy disease (MD) but are negative to genetic testing for FLT4. In this study, we aimed at performing a genetic characterization and biochemical analysis of VEGF-C variations found in a female proband born with congenital edema consistent with Milroy-like disease. METHODS: The proband underwent next-generation sequencing-based genetic testing for a panel of genes associated with known forms of hereditary lymphedema. Segregation analysis was performed on family members by direct sequencing. In vitro studies were performed to evaluate the role of a novel identified variant. RESULTS: Two VEGF-C variations were found in the proband, a novel p.(Ser65Arg) and a pathogenic c.148-3_148-2delCA, of paternal and maternal origin, respectively. Functional characterization of the p.(Ser65Arg) variation in vitro showed alterations in VEGF-C processing. CONCLUSIONS: Our findings reveal an interesting case in which biallelic variants in VEGF-C are found in a patient with Milroy-like lymphedema. These data expand our understanding of the etiology of congenital Milroy-like lymphedema.

Therapeutic Regeneration of Lymphatic and Immune Cell Functions upon Lympho-organoid Transplantation.

Lymph nodes (LNs) are secondary lymphoid tissues that play a critical role in filtering the lymph and promoting adaptive immune responses. Surgical resection of LNs, radiation therapy, or infections may damage lymphatic vasculature and compromise immune functions. Here, we describe the generation of functional synthetic lympho-organoids (LOs) using LN stromal progenitors and decellularized extracellular matrix-based scaffolds, two basic constituents of secondary lymphoid tissues. We show that upon transplantation at the site of resected LNs, LOs become integrated into the endogenous lymphatic vasculature and efficiently restore lymphatic drainage and perfusion. Upon immunization, LOs support the activation of antigen-specific immune responses, thus acquiring properties of native lymphoid tissues. These findings provide a proof-of-concept strategy for the development of functional lympho-organoids suitable for restoring lymphatic and immune cell functions.

Tolerance to cannabinoid response on the myenteric plexus of guinea-pig ileum and human small intestinal strips.

1. We studied tolerance to cannabinoid agonist action by comparing the in vitro inhibition of electrically evoked contractions of longitudinal muscle from small intestine of human and guinea-pig (myenteric plexus preparations) after 48-h incubation with the synthetic agonist (+) WIN 55,212-2. We also investigated the intrinsic response to the selective cannabinoid CB(1) receptor antagonist rimonabant in control and tolerant strips. 2. (+) WIN 55,212-2 inhibited guinea-pig (IC(50) 4.8 nM) and human small intestine (56 nM) contractions with similar potency before or after 48-h incubation in drug-free conditions; this effect was competitively antagonized by rimonabant (pA(2), 8.4, 8.2). A 48-h preincubation with (+) WIN 55,212-2, but not with (-) WIN 55,212-3, completely abolished the acute agonist response in both tissue preparations. The opiate K-receptor agonist U69593 inhibited human small intestine contractions with a similar potency in control and strips tolerant to (+) WIN 55,212-2, IC(50) 39 and 43 nM. 3. Unlike human tissue, in guinea-pig small intestine, which has a high level of endocannabinoids, rimonabant alone increased the twitches induced by the electrical field stimulation (EC(50) 100 nM) with a maximal effect of 123%. 4. In strips tolerant to (+) WIN 55,212-2, rimonabant markedly increased (155%) the electrical twitches in human ileum and in guinea-pig myenteric plexus smooth muscle (133%). 5. This study shows tolerance can be induced to the cannabinoids' action in intestinal strips of human and guinea-pig by long in vitro incubation with the agonist (+) WIN 55,212-2.

Transmural pressure during cardiogenic oscillations in rodent diaphragmatic lymphatic vessels.

BACKGROUND: The mechanism of initial lymphatic filling and the role of cardiogenic tissue motion in promoting lymph formation and propulsion are at present still controversial issues, in particular when considering interstitial tissues whose fluid pressure is well below atmospheric. To elucidate these aspects, the micropuncture technique was used to record interstitial (P(int)) and intralymphatic pressure (P(lymph)) simultaneously in the diaphragmatic lymphatic plexus draining the pleural cavity. METHODS AND RESULTS: The diaphragmatic lymphatic network was identified in anesthetized rabbits and rats through fluorescent dextrans injected intrapleurally. All P(lymph) and P(int) traces were pulsatile, oscillating either in-phase (33% of traces) or out-of-phase (67%) during cardiogenic swings. P(lymph) swept between -4.1 +/- 0.9 (SE) mmHg and 3.5 +/- 1.1 mmHg in rabbits, and between -5.1 +/- 1.0 mmHg and -2.7 +/- 1.1 mmHg in rats. P(int) oscillated between -0.8 +/- 0.7 mmHg and 4.9 +/- 0.7 mmHg in rabbits, and between -0.6 +/- 0.8 mmHg and 0.9 +/- 0.7 mmHg in rats. CONCLUSIONS: The data revealed a great functional complexity of the diaphragmatic lymphatic network and suggested that cardiogenic oscillations may play an important role in promoting lymph formation and propulsion from interstitial tissues with subatmospheric tissue pressure.

Development of functionally patent lymphatic meshes in postsurgical long-term resolution of peripheral secondary lymphedema.

OBJECTIVE: In oncologic surgery, secondary lymphedema of male external genital organs and upper or lower limbs frequently develops as a result of excision or mechanical obstruction of collecting lymphatic trunks. We evaluated whether the short-term and long-term outcomes of microsurgical treatment of limb and genital organs improves tissue drainage in patients with secondary lymphedema by restoring the pre-existing lymphatic networks or through new lymphangiogenesis. METHODS: Of 110 secondary lymphedema patients, microsurgery was performed in 45 hospitalized patients. Patients were aged 25 to 75 years, had at least third-degree lymphedema, no satisfactory results from previous physical or pharmacologic therapy, without primitive neoplasia, at least 1 year since the last postsurgical adjuvant oncological treatment, and <15 years since the previous primary oncologic lymphedema development. A microsurgical lymphovenous shunt of the spermatic cord (n = 7), a lymphovenous shunt of the lower limbs (n = 32), or lymphatic grafting of the upper limbs (n = 6) was performed. The male external genitals were treated through an innovative lymphovenous shunt of the lymphatic collectors in the pampiniform plexus of the spermatic cord. For lower limb lymphedema, the lymphatics were shunted to the collaterals or saphenous vein. For upper limb lymphedema, a shunt was performed between the lymph vessels of the jugular-supraclavicular area and those in close continuity with the axillary region. The patency of the new lymphatic pathways was assessed using Photodynamic Eye (Hamamatsu Photonic K.K., Tokyo, Japan) lymphography. RESULTS: Six months postoperatively, 36 responding patients showed an almost complete recovery from secondary lymphedema. Lymphatic meshes, consisting of several lymphatic vessels merging into well-canalized and complex networks developing in the perianastomotic area or between the adjacent proximal anastomotic lymphatic collectors, were commonly observed in patients who positively responded to microsurgery. These complexes were never encountered in nonresponding patients or in normal, nonedematous tissue. CONCLUSIONS: Long-term postsurgical recovery from severe secondary lymphedema requires canalizing the lymphatic collectors along their original flow pattern and developing perianastomotic meshes. Because this phenomenon can be observed with the same characteristics in different tissues, such as the spermatic cord and the inguinocrural, inguinoscrotal, inguinotesticular, and brachial regions, the development of meshes seems to reflect a generalized phenomenon of local lymphangiogenesis triggered by the microsurgical procedure.

Assessment and follow-up of patency after lymphovenous microsurgery for treatment of secondary lymphedema in external male genital organs.

Secondary lymphedema of external male genital organs is a frequent complication of pelvic radical surgery following pelvic lymphadenectomy. Microsurgical lymphovenous anastomoses are usually performed using only the superficial scrotal lymphatics, excluding testicular lymphatic drainage. We have experimented using a new microsurgical technique based on lymphovenous anastomosis between the collectors of the spermatic funiculus and the veins of the pampiniform plexus, allowing testicular lymphatic drainage. The study included 11 patients with external genital organ lymphedema, five of whom were subjected to microsurgical lymphovenous derivation. At 3, 6, and 12 mo after surgery, the patency of lymphovenous anastomoses was assessed by noninvasive lymphography using indocyanine green fluorescence images obtained with the Photodynamic Eye (PDE) infrared camera system (Hamamatsu Photonics K.K., Hamamatsu, Japan). Progressive improvement of clinical conditions was assessed both by patients' self evaluation and by objective clinical follow-up based on: (1) PDE lymphography, (2) tomography of the pubic area, (3) recovery of the soft consistency of the scrotal tissue, (4) recovery of the scrotal skin normochromic aspect, (5) absence of pain, and (6) disappearance of edema with evident reduction of the scrotal and penile dimensions and normal palpability of the testis. The present study shows that lymphovenous anastomosis is a valuable method of resolving the edematous condition. The indocyanine green approach for lymphangiography is a very supportive method during follow-up because, with the least invasive approach, it is possible to ascertain the complete patency of the anastomosis, to confirm its localization, and to assess its lymphatic drainage.

Secondary scrotal lymphedema: a novel microsurgical approach.

Secondary scrotal lymphedema is an infrequent complication of radical cystectomy assiociated with pelvic lymphadenectomy. We report a case of secondary lymphedema of male genitalia presenting more than 4 years after a radical cystectomy with extended pelvic lymphadenectomy for adenocarcinoma of the bladder. Microsurgical lymphovenous anastomoses are usually performed using only the scrotal lymphatics excluding the testicular lymphatics drainage. We have experimented a new microsurgical technique based on lymphovenous anastomosis between the collectors of the spermatic funiculus and the veins of the pampiniform plexus, allowing the testicular lymphatic drainage.

Transmural pressure in rat initial subpleural lymphatics during spontaneous or mechanical ventilation.

The role played by the mechanical tissue stress in supporting lymph formation and propulsion in thoracic tissues was studied in deeply anesthetized rats (n = 13) during spontaneous breathing or mechanical ventilation. After arterial and venous catheterization and insertion of an intratracheal cannula, fluorescent dextrans were injected intrapleurally to serve as lymphatic markers. After 2 h, the fluorescent intercostal lymphatics were identified, and the hydraulic pressure in lymphatic vessels (P lymph) and adjacent interstitial space (P int) was measured using micropuncture. During spontaneous breathing, end-expiratory P lymph and corresponding P int were -2.5 +/- 1.1 (SE) and 3.1 +/- 0.7 mmHg (P < 0.01), which dropped to -21.1 +/- 1.3 and -12.2 +/- 1.3 mmHg, respectively, at end inspiration. During mechanical ventilation with air at zero end-expiratory alveolar pressure, P lymph and P int were essentially unchanged at end expiration, but, at variance with spontaneous breathing, they increased at end inspiration to 28.1 +/- 7.9 and 28.2 +/- 6.3 mmHg, respectively. The hydraulic transmural pressure gradient (DeltaP tm = P lymph - P int) was in favor of lymph formation throughout the whole respiratory cycle (DeltaP tm = -6.8 +/- 1.2 mmHg) during spontaneous breathing but not during mechanical ventilation (DeltaP tm = -1.1 +/- 1.8 mmHg). Therefore, data suggest that local tissue stress associated with the active contraction of respiratory muscles is required to support an efficient lymphatic drainage from the thoracic tissues.