Cornelis J. M. Melief

Tumor eradication by adoptive transfer of cytotoxic T lymphocytes

Publisher Summary This chapter discusses the eradication of tumor by adoptive transfer of cytotoxic T lymphocytes. Complete and permanent eradication of tumors by histocompatability complex (MHC) class I restricted CD8 + tumor-specific cytotoxic T lymphocytes (CTLs) can be achieved in a variety of experimental murine models. In human metastatic melanoma, CD8 + tumor-specific CTLs are probably therapeutically the most active components among lymphokine-activated killer or tumor-infiltrating lymphocytes cells, with a possibly important contribution by CD4 + cells in some patients, although no clinical experience with cloned T cells exists as yet to evaluate this point. Cancer cells can avoid the induction of and destruction by tumorspecific CTLs in a number of ways, some of which can be manipulated to increase the efficacy of sensitization or adoptive therapy. T cell therapy allows targeting on minute changes in any cellular peptide that is presentable in the context of MHC molecules. CD8 + CTLs stand out as remarkably effective in tumor eradication in view of their remarkable potency, and specificity in allograft rejection and antiviral immunity.

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

SummarySix patients with progressive B cell non-Hodgkins lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15–30 mg. After the mAb infusions the number of circulating tumour cells was temporarily reduced, but in some cases antibody-coated cells remained in the circulation for several days. mAb penetrated to extravascular tumour sites; in general higher doses were required to saturate cells in the lymph nodes than to sensitize tumour cells in the bone marrow. mAb doses of up to 250 mg were given i.v. over 4 h without major toxicity. One patient twice achieved a partial remission after two periods of mAb treatment with an 8-month interval; the second remission lasted for 9 months. One patient showed a minor response. None of the patients made antibodies against the mouse immunoglobulin. Serum immunoglobulin levels were followed as a measure of the function of the normal B cell compartment; no significant changes were seen up to 6 months after mAb treatment.

Treatment of low-grade non-Hodgkin's lymphoma with continuous infusion of low-dose recombinant interleukin-2 in combination with the B-cell-specific monoclonal antibody CLB-CD19

Seven patients with low-grade non-Hodgkins lymphoma were treated with a combination of a murine monoclonal antibody directed against the B-cell-specific antigen CD19 (CLB-CD10), given twice weekly, and continuous infusion of low-dose recombinant interleukin-2 (rIL-2). We demonstrated stable serum CLB-CD19 levels throughout the 12 weeks of treatment, and homing of the antibody into the tumour sites. A variable degree of antigenic modulation was noted. Prolonged treatment resulted in a sustained increase in the number of natural killer cells in the circulation with enhanced cytotoxic capacity, including antibody-dependent cellular cytotoxicity. During the first weeks of treatment, T cell activation occurred in the majority of patients. Toxicity was related to the rIL-2 treatment and consisted of transient constitutional symptoms and a flu-like syndrome without organ dysfunction. A partial remission occurred in one patient, and in another patient who was primarily leukaemic a greater than 50% reduction of circulating B cells was noted. An antitumour effect occurred early during treatment and could not be related to rIL-2-induced modulation of natural killer cell or T lymphocyte activation.

CML typing of serologically identicalH-2 mutants

Cell-mediated lympholysis (CML) reactions were studied among four strains of C57BL/6 (B6) mice carrying mutant alleles (H-2ba,H-2bd,H-2bg, andH-2bh) at thez1 locus in theK end ofH-2b and the original B6 (H-2b) strain. Cross killing of target cells from lines that had not participated in the mixed lymphocyte reaction (MLR) was extensive, but usually less intense than that of target cells of stimulator cell genotype. The extent of CML crossreactivity could be limited by using cells from F1 hybrid mice as responders in MLR. In a comprehensive analysis of the cytotoxicity exerted by 20 MLR combinations with homozygous, and 10 MLR combinations with F1 hybrid responder cells, 19 different CML cytotoxicity patterns were identified, corresponding to at least 19 different CML target specificites. When the number of CML mismatches of each mutant with the originalH-2b was calculated,H-2ba was found to be most distinct fromH-2b,H-2bs andH-2bd were closest toH-2b, andH-2bh occupied an intermediate position. The validity of this sequence of relatedness is supported by published reports on skin graft survival times and on the interaction of T lymphocytes with virus-infected target cells using cells fromz1 locus mutants.

Enhancement of the antibody-dependent cellular cytotoxicity of human peripheral blood lymphocytes with interleukin-2 and interferon alpha.

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3−, CD56+, CD16+ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon α (rIFNα) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFNα induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15–30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20–50 U/ml. We now show that activation of the NK cells with a combination of rIL-2and rIFNα induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFNα was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of FcγRIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this FcγR was essential for ADCC of the human effector cells.

Alternative splicing in the mouse H-2Kd gene is not necessary for the classical Kd antigen function.

The mouse H-2Kd gene gives rise to several transcripts by alternative splicing. In addition to encoding the known Kd antigen, it could thus encode at least one minor hypothetical Kd-like molecule, with a distinct NH2 terminus. The existence of this “24” product can be inferred from a cDNA clone which was previously isolated. We have engineered both this cDNA and its canonical counterpart into a eukaryotic expression vector. After transfer of these constructs into mouse fibroblasts, we obtained cells expressing either one of the transcripts, but not both. In cytotoxicity tests, we found no expression of the “24” product on the cell surface, nor did we obtain any clue concerning its function. In contrast, cells which express Kd antigen, but none of the possible Kd-like molecules produced by alternative splicing, were functional in all aspects examined. We conclude that alternative splicing does not contribute to the known function of the Kd antigen.

Possible role for cytotoxic lymphocytes in the pathogenesis of acute interstitial nephritis after recombinant interleukin-2 treatment for renal cell cancer

A patient with renal cell cancer developed acute renal failure due to biopsy-proven acute tubulo-interstitial nephritis (AIN) in the 6th week of continuous infusion of 9 × 106 IU m−2 day−1 recombinant interleukin-2 (rIL-2). We investigated whether the AIN was the result of a cellular cytotoxic reaction induced by the rIL-2 treatment. The cytolytic activity of cryopreserved peripheral blood lymphocytes (PBL), isolated before and at the end of the rIL-2 treatment (at the time of AIN), was studied after 5 days of culture with or without rIL-2 or anti-CD28 and immobilized anti-CD3 antibodies. The PBL isolated before and at the end of the rIL-2 treatment showed cytolytic activity towards a number of allogeneic targets. However, only the PBL isolated at the end of the rIL-2 treatment showed, when stimulated with rIL-2 in vitro, significant cytolytic activity against an autologous renal cell line cultured from the AIN biopsy specimen and against an allogeneic renal cell cancer cell line. These PBL displayed no enhanced killing capacity towards autologous PBL and the melanoma cell line M14. These observations suggest that the AIN may be the result of a cytotoxic lymphocyte-mediated reaction induced by the rIL-2 treatment.

Different requirements for the regulation of transplantation tolerance induction for allogeneic versus xenogeneic major histocompatibility complex antigens

One way to solve the problem of human donor organ shortage is the use of animal organs. Therefore, it is important to study the T-cell response against xenogeneic major histocompatibility complex (MHC) antigens. In the present study, we have used HLA-B27 transgenic mice in a xenogeneic transplantation model. The results indicate that both transgenic skin transplantation and intravenous (IV) injection of transgenic spleen cells can reverse specific T-cell low responsiveness against the transgenic HLA-B27 antigen into high responsiveness in vivo and in vitro. In contrast, IV injection of spleen cells across an allogeneic H-2 class I disparity results in transplantation tolerance. Thus, despite T-cell low responsiveness against the transgenic HLA-B27 antigen, IV injection of transgenic HLA-B27 disparate lymphocytes does not tolerize, but rather immunizes for the xeno-MHC antigen.

Remission of monoclonal chronic T-cell expansion associated with severe anemia

A 49-year-old male with an 8 year history of lowered Hb level, granulocytopenia and fatigue presented in 1986 with progressive fatigue, a dramatically reduced Hb level (45 g/l) and an increased lymphocyte count (6.6 x 10(9)/l). Clinical picture and laboratory studies led to the diagnosis of chronic T lymphocytosis with expansion of CD8+ T cells expressing CD16 IgG Fc receptors (Fc gamma RIII). DNA analyzed with T-cell receptor (TcR) gamma and beta probes revealed extra rearranged bands representing a clonal expansion of T lymphocytes. These T lymphocytes expressed T-cell receptor alpha beta as evaluated by staining with monoclonal antibodies. Because of the severe progressive anemia the patient was transfused with packed red cells. He was then treated with cyclophosphamide. After one month of treatment the transfusions could be discontinued and two months later cyclophosphamide treatment was stopped because of normalized Hb level and lymphocyte counts. The patient remained in a hematologically stable condition, though a minor T-cell population representing the clonal expansion, an inverted CD4/CD8 ratio and low immunoglobulin levels persisted. This is the first report of regression of proven monoclonal CD8+ T gamma-cell expansion and the associated anemia following cyclophosphamide therapy. These observations implicate the expanded monoclonal CD8+ lymphocytes in the pathogenesis of the anemia and granulocytopenia.

Genetic Control of T-Cell and NK-Cell Protection Against Lethal Sendai Virus Infection

The in vivo importance of class I MHC regulation of the cytotoxic T cell (Tc) response to a natural pathogenic agent of high virulence was studied on the basis of our demonstration of a major difference in the capacity to generate a Sendai virus-specific Tc response between C57BL/6 (B6, H-2b) mice and H-2Kb mutant B6.C-H-2bm1 (bm1) mice. These two mouse strains differ from each other only in three amino acids in the crucial H-2Kb restriction element for this response. Bm1 mice, in contrast to B6 mice, are Tc nonresponders against this virus, but show Sendai-specific T cell proliferation, antibody production, and delayed type hypersensitivity (DTH) reactions, as well as natural killer (NK) cell activity, equal to those of B6 mice. Another H-2b mouse strain, the 129/J, also shows equal Sendai virus-specific Tc, T helper cell (Th), B cell and DTH responses compared to B6, but is virtually deficient in generating an NK cell response. B6, Sendai Tc-deficient bm1, T cell-deficient B6 nu/nu and NK low-responder 129/J mice differ from each other in susceptibility to lethal pneumonia induced by intranasal (i.n.) inoculation of virulent Sendai virus. The lethal dose (LD50) in B6 mice averages 152 TCID50, in bm1 mice 14 TCID50, in B6 nu/nu mice 0.5 TCID50 and in 129/J mice 0.2 TCID50. The importance of Tc is shown by the difference in susceptibility between B6 and bm1 mice and also by the complete protection of B6 nu/nu mice against infection with a lethal virus dose by i.v. injection of a Sendai virus-specific, IL-2 dependent and H-2Kb restricted B6 Tc clone. In vivo protection by this Tc clone is H-2Kb restricted. Apart from Tc, an important role for virus-specific Th cells is evident from the difference in susceptibility between bm1 and B6 nu/nu mice. This conclusion is supported by the demonstration that the mean survival time of B6 nu/nu and bm1 nu/nu mice can be significantly prolonged, in an I-Ab restricted manner, by the injection of in vitro-propagated, Sendai-specific B6 or bm1 Th clones after a lethal dose of Sendai virus, and by the demonstration that inoculation of these Th clones provides help to virus-specific Tc by means of IL-2 production. Strikingly, Th and Tc cooperate in anti-Sendai virus immunity, since permanent survival of lethally infected nu/nu mice is only achieved by inoculation of a mixture of Tc and Th clones or a mixture of a Tc clone and rIL-2. Furthermore, the difference in susceptibility to Sendai virus infection between B6 and bm1 mice provides a unique model for the study of MHC-disease associations. The importance of NK cells is revealed by the high susceptibility of 129/J mice to Sendai virus infection, although all other immune parameters measured appear to be normal.