Cristina Jemma

Treatment of recurrent squamous cell carcinoma of the head and neck with low doses of lnterleukin‐2 injected periiymphatically

Ten patients with recurrent squamous cell carcinoma of the head and neck received daily injections of interleukin‐2 (IL‐2) from the Jurkat T‐cell line purified by high pressure liquid chromatography for 10 days. Two hundred units of IL‐2 in 0.5 ml were injected 1.5 cm from the insertion of the sternocleidomastoid muscle on the mastoid. When possible, courses were repeated at 45‐day intervals. IL‐2 was ineffective in two patients who had already undergone functional or radical neck dissection. By contrast, in six patients with contralateral or bilateral cervical lymph nodes, complete or partial disappearance of the tumor was observed. The injections were occasionally followed by moderate local swelling and lymph node pain, but no systemic disturbances.

Helper strategy in tumor immunology: expansion of helper lymphocytes and utilization of helper lymphokines for experimental and clinical immunotherapy.

Two main kinds of immune strategy are possible against neoplasia. The first potentiates a selected effector arm. In vitro culture with exogenous interleukin-2 (IL-2) increases the activity of natural killer cells and leads to the expansion of T cytotoxic lymphocytes. Systemic reinfusion of both of these cells with high doses of IL-2 mediates the regression of a variety of murine and human tumors. In an alternative strategy, a few regulatory lymphocytes turn on immune reactivity by triggering a cascade of interconnected effector functions. The efficacy of this strategy rests on the repertoire of effector mechanisms moved to action. An effective immunoregulatory maneuver is the addition of helper determinants on the surface of tumor cells. Its power can be further increased by the pre-induction of helper T lymphocytes specific to the helper determinants. This approach can be achieved in mice by coupling muramyl dipeptides to tumor cells, along with eliciting T lymphocytes specifically reactive to Bacillus Calmette-Guerin. Noncytotoxic T helper lymphocytes produce factors which recruit nonspecific (macrophages) as well as specific (cytolytic T lymphocytes) anti-tumor attacking cells. In this way protection can be afforded against primary tumors and metastases, as well as leukemia cells. As the activity of helper lymphocytes rests mostly on lymphokine release, the use of molecularly defined lymphokines mimicking T-helper functions has also been attempted. In a few experimental models, the association of low doses of IL-2 with non-reactive lymphocytes from tumor-bearing mice promotes an effective anti-tumor reaction in the host. Moreover, the combination of distinct lymphokines can also build a molecularly defined helper system able to activate in sequence non-specific and specific anti-tumor reactions in vivo. Trials intended to evaluate the clinical impact of these helper approaches in the management of human tumors are being started or are already under way.

Temporary regression of recurrent squamous cell carcinoma of the head and neck is achieved with a low but not with a high dose of recombinant interleukin 2 injected perilymphatically

The efficacy of ten daily injections of 500 or 500,000 U of recombinant interleukin 2 (IL-2) day-1 given 1.5 cm from the insertion of the sternocleidomastoid muscle on the mastoid was evaluated in 31 patients with recurrent head and neck squamous cell carcinoma. No toxic effects were noted. One complete response (CR) and three partial responses (PRs) were observed in the 16 patients who received 500 U of IL-2, whereas the higher dose was not effective. The CR was recorded in one of the seven patients with a oropharyngeal recurrence. Partial responses were obtained in 1/5 patients with hypopharyngeal recurrences, in 1/5 patients with oral cavity recurrences and 1/7 patients with laryngeal recurrences. The duration of the responses was 3-5 months and additional courses of ten injections of IL-2 had no further effect.

The Effect of Preoperative Local Interleukin-2 (IL-2) Injections in Patients with Head and Neck Squamous Cell Carcinoma: An Immunological Study

Clinical, immunological, immunophenotypical, pathological and molecular biological studies were performed on tumor infiltrating lymphocytes (TIL) and lymph node lymphocytes (LN-ly) of 8 patients with squamous cell carcinoma of the oral cavity and oropharynx treated with 10 daily locoregional injections of low doses of IL-2 before surgery. No complications were seen during or after surgery. In 3 cases the LN-ly showed a moderate LAK activity, higher in the LN-ly omolateral to the tumor and near the IL-2 injection site; in 2 of these 3 patients a good LAK activity was induced after 6-day culture with IL-2. The LN-ly derived from nodes next to the tumor showed a decreased NK activity and proliferative ability both in basal conditions and after in vitro lymphokine challenging. LN-ly of 2 IL-2 treated patients showed high levels of mRNA encoding for IL-2-R, while it was absent in 2 untreated cases. Immunophenotypical studies on TIL showed statistically improved levels of CD25+ and LAK1+ cells in treated cases. Clusters of CD11c+ (macrophages) cells were seen close to the neoplastic sheets.

In vitro and in vivo comparison of the activity of human lymphokine-activated killer (LAK) cells and adherent LAK cells

Summary: The effectiveness of “standard” lymphokine-activated killer (LAK) cells, recovered after 6 days, and of “standard” adherent LAK (ALAK) cells, recovered after 14 days of culture in the presence of recombinant interleukin-2 (rIL-2) from peripheral blood lymphocytes of 21 healthy donors, was assessed through comparison of their proliferation, surface markers, cytotoxic activity, lymphokine production, and antitumor activity. In the presence of rIL-2, plastic adherent precursors of A-LAK cells proliferated much better than those of “standard LAK” cells and expanded even more than 300-fold. However, the final cell recovery of A-LAK was always lower because of their very few precursors, and the total lytic units (LUs) generated in A-LAK cultures were always lower for the same reason. On the other hand, the lytic activity of each A-LAK cell was always higher than that of a LAK cell. This was particularly evident on day 6 of culture. Removal of nonadherent cells after the first 24 h culture resulted in a significant enrichment in CD3¯CD56+ and CD8 + CD56+ cells in A-LAK cells, with a marginal number of CD4+ cells. A significant direct correlation between LUs and A-LAK CD3¯CD56+ percentage was found. In the presence of rIL-2, A-LAK cells produced higher amounts of tumor necrosis factor-α and interferon-γ than LAK cells, while only A-LAK cells produced IL-iβ and small amounts of IL-4. Neither LAK nor A-LAK produced IL-2. In the absence of injections of IL-2, LAK and A-LAK cells were equally able to inhibit the growth of a human T-cell lymphoma in immunosuppressed nude mice.

Serial Transplantation of a Human Acute T Lymphoblastic Leukemia into Nude Mice

A human acute T lymphoblastic leukemia line (PF-382) was serially transplanted into nude mice. No takes were observed in untreated nude mice, whereas solid tumors were observed in splenectomized and total body, sublethally irradiated mice. The minimal tumor-inducing dose and the latency time remained unchanged after the third and fifth serial transplants. Moreover, leukemic cells recovered from the 8th in vivo passages displayed the same differentiation antigens and chromosomal markers as the in vitro PF-382 cell line used for the first transplant. This stable and well-characterized experimental system could be a new model for T-lymphocyte differentiation and immune-reactivity against human leukemias.

In vitro and in vivo immunomodulatory activity of an N-9 arginyl hypoxanthine derivative (PCF-39).

A new synthetic derivative, N-alpha-5 (1,6-dihydro-6-oxo-9-purinyl) pentyloxy-carbonyl-L-arginine (PCF-39) has been evaluated in vitro and in vivo in order to clarify its immunopharmacologic profile. In vitro, PCF-39 did not modify spleen cell functions, whereas parenteral administration in mice of 2.5 and 25 mg/kg (50 and 500 micrograms/mouse) induced an increase in spleen and lymph node cellularity that resulted in a significant resistance to the growth of two distinct syngeneic transplanted tumors. These in vivo findings show that PCF-39 is a potent immunotherapeutic agent with an antitumor effect.

Functional properties in Sézary cells with an unusual phenotype

The immunological and functional characteristics of Sézary cells with an unusual phenotype are reported. The clinical, histologic, and hematologic picture was typical for Sézary syndrome. Studies with monoclonal antibodies showed that 80% Sézary cells had an CD3+, CD4+, CD5+, CD7-, CD8-, Leu-7+, Leu-8-, Leu-11-, OKM1- phenotype. By two-color immunofluorescence assay 80% FACS-sorted Leu-7+ cells coexpressed CD4 antigen and did not express the myeloid antigen OKM1, CD8, and antigens characteristic of immature T cells. The cells had no NK activity but did display a high helper activity. Unseparated and FACS-sorted Leu-7+ and Leu-7- Sézary cells did not respond to mitogens but were able to grow in the presence of exogenous IL-2. FACS sorted Leu-7- cells, cultured for 7 days in the presence of 20% IL-2, acquired the receptors for Leu-7. IL-2 and IFN-gamma production was studied in unseparated Leu-7+ and Leu-7- FACS-sorted Sézary cells. IL-2 production was lower than in normal cells. The addition of PHA or PHA plus TPA led to an increase in IL-2 production. Also IFN-gamma production was marked lower than in normal controls but increased after 7-day culture in exogenous IL-2. In conclusion in this case the Sézary cells may represent a neoplastic expansion of the CD3+, CD4+, CD5+, Leu-7+, Leu-11- subpopulation which is equivalent to the 2-4% of the Leu-7+ population in normal lymphocytes.

IN SITU HYBRIDIZATION EVIDENCE OF THE DONOR ORIGIN OF A POST-TRANSPLANT LYMPHOPROLIFERATIVE DISORDER

To the Editor: Post-transplant lymphoproliferative disorders (PTLPDs) are regarded as a group of EpsteinBarr virus (EBV)-driven lymphoid proliferations of varying clonal composition (1, 2). Studies of their genetic origin have shown that they may arise from both autologous and donor lymphocytes, depending on the tissue or organ transplanted. Although host cells are the most common source of PTLPDs arising in solid organ recipients (3, 4), a few cases originating from donor lymphocytes have been recorded (5-14). Here we describe the case of a 60-yr-old woman who underwent orthotopic liver transplantation because of liver failure due to chronic hepatitis C infection. The donor was an 18-yr-old boy who had died in a road accident. Serological screening before explanation revealed previous exposure to cytomegalovirus and hepatitis A virus. EBV exposure was assessed by a standard indirect immunofluorescence procedure detecting antibodies to EBV viral capsid antigen: a 1:80 titer of VCA IgG was found. The post-transplantation course was uneventful, and the patient was discharged on the 24th day with a combined immunosuppressive regimen including cyclosporine, azathioprine and methylprednisolone sodium succinate. Five months later she was readmitted with fever. Abdominal ultrasonographic examination revealed a focal solid lesion in the liver. An angio-CT scan was consistent with the presence of a lymphoproliferative lesion, and biopsy showed that this was a B-cell lymphoma. The patient underwent hemiepatectomy with total resection of the neoplastic mass. An intraoperative bone marrow biopsy disclosed a hypocellular bone marrow with no neoplastic infiltration. Histological examination on formalin-fixed paraffin-embedded material showed that the lesion consisted of large atypical lymphoid cells with irregular nuclei and one or more large nucleoli, intermingled with small lymphocytes. Residual sheets of hepatocytes were sometimes entrapped in the neoplastic component. Immunohistochemical tests on the neoplastic cells showed they were strongly positive for Bcell markers such as CD19 and CD20, whereas T-cell antigens were expressed by the intermingled small lymphocytes; surface Ig lightchain expression showed a clear-cut K-chain restriction, reflecting monoclonal expansion of B-cells. This feature was supported by Southern blot analysis of the Ig heavy-chain locus with a JH probe that demonstrated the presence of a single rearranged Ig heavy-chain band. According to the histopathological criteria described by Frizzera et al. (1) and modified by Knowles et al. (2), the lesion was diagnosed as a polymorphic large B-cell lymphoma. The patient underwent conventional polychemotherapy and, to date, is well with no evidence of disease after 3 yr follow-up. The development of a PTLPD in an allografted organ raises the question whether it originates from lymphoid cells donated with the transplanted organ, or from the recipient’s own lymphocytes. Another question concerns the role of EBV-host interactions during immunosuppression in the process of lymphomagenesis. To address this last point, namely whether viral infection occurred as a passenger event due to the immunocompromised status, or was more directly involved in the neoplastic proliferation, we investigated the cell localization and clonality of the EBV genome by in situ hybridization (ISH) for m-RNAs (EBER) on embedded sections of the liver tumor, and Southern blot (SB) analysis using a molecular probe detecting clonally expanded fused termini of circularized EBV episomes (15). The ISH was strongly positive in most lymphoma cells, indicating the presence of a latent EBV infection, whereas detection of a single EBV episomal band was consistent with the origin of the lymphoma from a single EBV-

Tumor Immunotherapy with Combined Interleukins Injected Perilymphatically: Experimental and Clinical Findings

Tumor growth is often perceived by the immune system. Weak cellular reactivity can be shown in the early stages, both in man and in experimental models. In preimmunisation experiments in mice, the immune system has proved capable of inhibiting syngeneic tumor growth1, while lymphocytes from cancer patients have been shown to react with autologous tumor cells in vitro in proliferation and cytotoxicity assays2,3. Nevertheless, most spontaneous or transplanted tumors grow and kill their host. Tumor cells can directly suppress host reactivity by secreting soluble mediators, and actively trigger specific or non-specific suppressor mechanisms that block both natural and adaptive host resistance4.