Search

We performed a retrospective study of the infectious complications in 14 pediatric patients undergoing transplantation with T lymphocyte-depleted bone marrow. The purpose of this study was to document the infectious complications encountered in these patients and to ascertain whether or not they appeared to be more susceptible to any particular infections. It was found that all 14 patients developed infectious complications following transplantation: there were a total of 24 bacterial infections among 11 patients, 21 fungal infections among 11 patients, 2 parasitic infections and 4 viral infections. The viral infections included two cases of infection with cytomegalovirus, one of which manifested as encephalitis and pneumonia, and one case of possible infection with Epstein-Barr virus, which included clinical findings compatible with a lymphoproliferative disorder. These patients were at an increased risk to certain viral infections and particular attention must be paid to the prevention, diagnosis and treatment of these infections.

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.

Early graft rejection crises in renal transplant patients pretreated with donor-specific transfusions (DST) have been attributed to an antibody-mediated reaction, or an in situ primed lymphocyte test (PLT) reaction in the grafted kidney resulting from priming of recipient T cells. To test these hypotheses, we analyzed the mixed lymphocyte culture (MLC) response to donor stimulator cells in 18 DST + azathioprine (AZA)-treated patients, including 7 with living-nonrelated donors (2 HLA-haplotype mismatch) and 11 with living-related donors (1 HLA-haplotype mismatch). Studies of the kinetics of anti-donor MLC responses of PBL obtained from patients before and after DST + AZA treatment revealed shifts in the magnitude and timing of antidonor MLC response. We found an increased peak MLC response to donor and/or third-party in 6/18 patients, 4 of whom had early rejection crises; the other 2 patients had blocking factors in their post-DST plasma that strongly (57-71%) inhibited their MLC response. Plasma factors were implicated in the mechanism of early graft crisis, in that patients with an enhanced MLC response in the presence of post-DST plasma showed a significantly (P less than .02) shorter time to first rejection episodes as compared with patients with plasma blocking factors. Finally, we confirmed the findings of others that a significant proportion (7/18) of DST + AZA-treated patients had a decreased MLC response. Although such patients experienced classic (2nd week or later) rejection episodes, none had a DST-type (less than or equal to 3 day) crisis. Our data supports the concept of T cell priming by DST, and suggests that patients who fail to develop concomitant suppressor cells or humoral blocking factors will develop a rapid onset cellular rejection crisis in the transplanted kidney.

Eleven patients received four consecutive weekly cycles of human recombinant interleukin 2 (IL-2) by continuous infusion for 4 days/week. Two dose levels were tested, 1 and 3 X 10(6) units/m2/day. Toxicities experienced by most patients included fever, rigors, fatigue, anemia, eosinophilia, and liver function abnormalities. All side effects from treatment reversed and no severe or life-threatening problems occurred. A marked lymphocytosis was seen following the 4 weeks of therapy. Fresh lymphocytes obtained during this lymphocytosis mediated enhanced destruction in vitro of a natural killer cell-resistant tumor cell line (Daudi). The increase in the absolute number of circulating lymphocytes and their enhanced ability to mediate direct lysis of Daudi targets resulted in a greater than 100-fold mean increase in cytotoxic potential by the end of IL-2 treatment. One patient, with renal carcinoma, who was treated at 3 X 10(6) units/m2/day experienced a sustained measurable response with greater than 50% regression of pulmonary and hepatic metastases. Five patients were retreated with a second course of IL-2, lasting 4 weeks. This therapy was well tolerated in four of these five patients, with similar immunological changes occurring. No further antitumor responses were seen in these patients. Thus, a relatively well tolerated immunotherapy regimen using IL-2 can induce dramatic increases in lymphocyte number and augment their in vitro antitumor reactivity.

In vitro sensitized cytotoxic T lymphocytes (CTLs) primarily detect antigens controlled within the major histocompatibility complex (MHC) that are associated with the serologically detected (SD) loci LA and FOUR. Frequently CTLs sensitized to cells from one individual kill target cells of a third party that shares no SD cross-reactivities by serological criteria with the initial stimulating cells. We have studied the possibility that this cross-killing is due to the recognition of non-MHC controlled antigens. Allogeneic tissue from MHC identical siblings is rejected in the absence of immunosuppression, demonstrating the importance of non-MHC antigens. Nevertheless, CTLs sensitized simultaneously with mitomycin-C treated cells from an MHC identical sibling and from an unrelated individual were only able to mediate cytotoxicity on the unrelated's target cells. No significant cytotoxicity was observed in any combination on target cells differing only for non-MHC antigens.

The immunomodulatory effect of two highly purified recombinant human interferons (IFN) on T cell-mediated cytotoxicity of autologous Epstein-Barr virus-transformed B cells (EBV-LCL) was assessed. Interferon beta ser, but not alpha 76, potentiated the destruction mediated by interleukin-2 (IL-2) dependent, long-term T cell cultures. The degree of potentiation of specific cytotoxicity was dependent on the concentration of the exogenously added IFN beta ser. In contrast to the potentiation of T cell-mediated cytotoxicity, IFN beta ser and alpha 76 did not modulate the cytotoxic activity of cytotoxic T cell clones that are specifically reactive to autologous EBV-LCL. These results indicate that the selective potentiation of EBV-LCL-reactive T cell-mediated cytotoxicity by IFN beta ser may not be due to a direct IFN effect on the cytotoxic T cells (CTLs) themselves. Rather, our results are consistent with IFN beta ser augmenting cytotoxic T cell reactivity indirectly, possibly via its influence on other immunoregulatory cells. Although the precise mechanism whereby IFN beta ser may potentiate cytotoxicity is not yet defined, the synergistic action of IFN beta ser and IL-2 on long-term CTL cultures offers an alternate means to selectively enhance a desirable cytotoxic response.

A single patient who had a leukemic relapse six months after receiving a syngeneic bone marrow transplant was given "adoptive chemoimmunotherapy." Lymphocytes from the patients identical twin were alloactivated and grown in T cell growth factor in vitro. After receiving chemotherapy to reduce the number of relapsed leukemia cells, he received 1.1 X 10(10) in vitro alloactivated twin lymphocytes via intravenous and intraperitoneal injections. Although progressive Candidal pneumonia was fatal and prevented analysis of either efficacy or delayed toxicity of this potential form of therapy for this patient, radioactive 111In labelling and scanning showed dissemination of these lymphocytes following either injection route, with no clinical evidence of immediate toxicity.

Ongoing clinical and laboratory research is being directed at the many problems and complications associated with clinical bone marrow transplantation. The most important goals are to increase the long-term survival of patients receiving bone marrow transplants, decrease the morbidity associated with the process, and extend this therapeutic modality to patients in need of a transplant, but without an HLA identical sibling. In addition, as these problems are resolved, and the toxicity of BMT is controlled, this form of therapy may become the treatment of choice for a number of inherited and acquired hematologic, immunologic, and metabolic disorders that are chronically progressive but ultimately fatal, yet are not now routinely treated with BMT due to the acute morbidity and mortality associated with this major procedure.

The availability of purified human recombinant interleukin 2 (IL-2) has enabled clinical trials to test its in vivo effects. We report here the immunological effects of 7 consecutive days of IL-2 treatment given to 25 patients with cancer in a clinical Phase I study. Peripheral blood lymphocytes obtained from patients following therapy with IL-2 had enhanced proliferative responses to IL-2 and enhanced direct cytotoxic activity on K562 target cells. This lytic activity was further augmented by the addition of IL-2 during the 51Cr release assay. Fresh peripheral blood lymphocytes from some patients who had just completed treatment at the higher IL-2 dose levels were able to kill both the natural killer-resistant Daudi cell line and fresh tumor cells while pretreatment samples and peripheral blood lymphocytes from healthy controls were not. This lytic activity was best detected when IL-2 was present in the in vitro effector assay. These results demonstrate that the administration of IL-2 to patients with cancer induces a population of effector cells able to directly destroy natural killer-resistant target cells, when assayed in the presence of IL-2.

A total of 41 patients with hematologic malignancies (other than acute leukemia in relapse) received allogeneic bone marrow transplants at the University of Wisconsin from 1 April 1980 through 31 March 1984. In an effort to minimize graft-versus-host disease, marrow was depleted of T-lymphocytes in vitro with monoclonal anti-T-cell antibody and complement prior to infusion for seven of 19 recipients of marrow from HLA-identical, MLC-nonreactive siblings, and for all 22 recipients of marrow from MLC-reactive HLA-haploidentical donors. The recipients of HLA-identical T-depleted marrow all showed excellent engraftment following standard pre-BMT conditioning with cyclophosphamide and total body irradiation. In contrast, five of five recipients of T-depleted haploidentical marrow failed to engraft following this same conditioning regimen. The addition of cytosine arabinoside to the pretransplant conditioning appeared to correct this problem, allowing engraftment in 14 of 17 subsequent patients. These clinical results, coupled with prior in vitro data, demonstrate the need to adequately suppress residual host-versus-graft immunity in order to prevent the rejection of T-cell-depleted HLA-haploidentical bone marrow.

We have utilized serological techniques and mixed lymphocyte culture (MLC) reactions to examine HLA-DR and HLA-D expression by heated (45 degrees C for 1 h) lymphocytes in order to study the functional relationship of these antigens. Heated lymphocytes do not stimulate proliferation of allogeneic lymphocytes in MLC, yet they express HLA-DR antigens. The fraction of peripheral blood lymphocytes (PBL) expressing DR is not altered by heating, nor is the staining intensity altered as detected by fluorescence microscopy. Alloantisera to "B cell alloantigens" recognize HLA-DR determinants on heated cells without any detectable change in either specificity or quantitative cytotoxic effects. Flow cytometry with monoclonal antibody demonstrates only minimal decrease in HLA-DR expression after heating. Thus stimulation in MLC requires more of the stimulating cell than the mere expression of HLA-DR.

The human Epstein-Barr virus transformed lymphoblastoid cell line (EBV-LCL) 721 and MHC haplotype loss variants derived from it were utilized to dissect the functional role of MHC genes in the proliferative response of autologous T lymphocytes to EBV-LCL. LCL-721 is heterozygous at all phenotypically defined MHC loci. One type of LCL-721 variant expresses only determinants encoded by the maternal (m) haplotype and the other expresses determinants encoded by the paternal (p) haplotype. Autologous (individual A) primary proliferative responses are strong to each type of haplotype deletant. The strong tertiary responses to the priming haplotype in comparison to the relatively weak responses to the reciprocal haplotype indicate that MHC linked genes encoded by each haplotype are important in the autologous response to EBV-LCL. Similar specific tertiary responses are observed when peripheral blood lymphocytes (PBLs) from the donor's mother are used as responding cells. Allogeneic responses were also studied by priming PBLs from unrelated donors with the haplotype deletants. Quantitative comparisons of the proliferation by primed allogeneic and autologous lymphocytes stimulated by irradiated PBLs from donor A and her mother, and by LCL-721 and its variants, show that some of the tertiary responses involve specific recognition of EBV-LCL while others detect recognition of alloantigens.

Cells other than the macrophage can function as antigen-presenting cells (APCs). These class II-bearing accessory cells include dendritic cells, epidermal Langerhans cells, B cells, murine B-cell tumors, and human Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL). We investigated the ability of EBV-LCL to present two soluble antigens, Candida albicans and purified protein derivative of tuberculin (PPD). The EBV-LCL derived from B cells of two different individuals can present both antigens to bulk cultures of autologous antigen-primed peripheral blood lymphocytes. The responses of PPD-reactive T-cell clones were weaker to PPD when presented by EBV-LCL than by PBL-APCs, with some clones responding only to PPD presented by PBL-APCs. This suggests that EBV-LCL are not equivalent to PBL monocytes in APC function, and that expression of class II major histocompatibility complex antigen is not sufficient in enabling antigen-presenting capability.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality following bone marrow transplantation. The in vitro removal of the GVHD-causing T-lymphocytes from donor marrow is one approach which could control this complication. Treatment of the donor bone marrow with lectins and erythrocyte-forming rosette depletion, anti-T-cell antisera or monoclonal antibodies are methods currently being tested to accomplish this. CT-2 is an immunoglobulin monoclonal antibody specific for the T-cell erythrocyte-forming rosette receptor. Bone marrow from 23 consecutive donors was treated in vitro with CT-2 and complement, prior to infusion, as a potential means of controlling GVHD. Surface marker analysis using erythrocyte-forming rosetting, and OKT-3 and OKT-11 monoclonal antibodies on paired samples of treated and untreated marrow demonstrated a mean depletion to 1% of the original number of T-cells. Proliferative responses to alloantigens and mitogens as well as cytotoxic and natural killer cell function were tested and found to be markedly reduced. Despite these effects on T-lymphocytes, viable hematopoietic stem cell colonies were retained. Clinical results following the in vitro T-lymphocyte depletion of donor bone marrow for the 8 histocompatible and 15 nonhistocompatible bone marrow transplantation are reported. Prompt engraftment with minimal GVHD, despite no posttransplant GVHD prophylaxis, was seen in seven of the matched patients. In the nonhistocompatible bone marrow transplantation, failure of engraftment occurred in 11 patients. Grades III-IV GVHD were seen in two of the four patients that engrafted despite good T-lymphocyte depletion. No predictive correlation could be found between the in vitro analysis of marrow following CT-2 treatment and clinical outcome.

Epstein-Barr virus-transformed human B lymphoblastoid cell lines (EBV-LCL) can present soluble antigens to antigen-primed T lymphocytes. In this study, we used HLA antigen-loss mutants of an EBV-LCL line (LCL 721) to demonstrate that the presentation of a soluble antigen from Candida albicans (CAN) by EBV-LCL to primed T cells can be restricted by multiple HLA determinants. Haplotype-deletion mutants that contained only the maternal or only the paternal HLA-haplotype were used to demonstrate the preferential role of autologous HLA antigens in presenting soluble antigens to Candida-primed T cells from the donor of LCL-721, and to T cells from her mother and father. Immunoselected mutants of LCL-721 showing a variety of distinct phenotypes that are deficient in HLA-DR, DQ, or DP antigen expression were tested as antigen-presenting cells. The antigen-presenting ability of these class II deficient EBV-LCL variants weakened with progressive loss of class II HLA determinants expressed on the cell surface. Our study, therefore, provides evidence for multiple HLA restriction determinants, including HLA-DR, DQ, and DP. Furthermore, LCL lacking all HLA-DR, DQ, and DP expression because of homozygous deletion of these MHC class II genes still presented CAN and Tetanus toxid (TET), although to a much lesser degree than presented by LCL-721. This suggests that determinants other than DR, DQ, and DP which are expressed on these EBV-LCL may also function as restriction elements for the proliferative T-cell response to soluble antigens.

Immune stimulation or interferon administration induces indoleamine 2,3-dioxygenase and GTP cyclohydrolase activity in humans, resulting, respectively, in tryptophan degradation to kynurenine and in neopterin production. To determine if similar effects result from interleukin 2 (IL-2) administration, plasma tryptophan and urinary kynurenine and neopterin were measured in patients undergoing a phase 1 toxicity trial of recombinant IL-2 given by daily bolus or continuous i.v. administration for 7 days at doses of 1 x 10(5) to 1 x 10(7) units/m2/day. Significant dose-dependent decreases in plasma tryptophan levels and corresponding increases in urinary kynurenine and neopterin were observed. These metabolic effects of IL-2 are probably mediated by induction of gamma-interferon production, although elevated levels of gamma-interferon were not found in the sera of these patients. In view of the indispensable role of tryptophan in synthesis of protein, niacin, and serotonin, we suggest that some of the toxic side effects may be the result of this loss of tryptophan. Since these metabolic changes were detected at relatively low doses of IL-2, these assays provide a highly sensitive means for monitoring in vivo metabolic responses to IL-2 therapy.

We have presented the rationale for the in vitro approaches that we have taken for generating cytotoxic lymphocytes capable of lysing autologous leukemia cells or leukemia cells from HLA identical siblings. Two different approaches have been used, both of which are based on earlier findings concerning the antigenic and cellular interactions involved in the generation of strong cytotoxic responses to alloantigens in mixed leukocyte culture.

Despite the abundance of evidence from murine models suggesting a powerful immunological approach to the treatment of cancer, the available data on clinical response have not been as dramatic. The use of interleukin-2 (IL-2) either alone or in combination with lymphokine activated killer (LAK) cells clearly is therapeutic for some patients, albeit a minority. It does not appear from the available evidence that the regimens tested show major differences in antitumour activity, although there is a sense that higher doses may be slightly more effective. Nor is it clear that the addition of LAK cells significantly or sufficiently enhances clinical responses to warrant their widespread use. It must therefore be concluded that other patient and tumour related factors must have an undefined role in the ability to attain meaningful responses. Immunological response to IL-2 and tumour burden are factors which can be examined given the available clinical data. While animal studies have shown that antitumor effects are related to the dose and number of LAK cells given, this is not as clear in patient studies. Some reports have suggested a correlation between clinical response and the in vivo induction of LAK activity or the magnitude of the rebound lymphocytosis. Clinical trials at the University of Wisconsin have shown striking increases in both the number of peripheral blood lymphocytes and LAK induction in patients who showed no clinical response. This in vivo LAK induction, as expected, is not the sole determinant in achieving a measurable response. Whether it is a necessary biological response needed to achieve antitumour effects remains uncertain. The role of tumour burden in response to IL-2 remains elusive. In many trials, including our own, patients with bulky disease have responded to therapy that was ineffective in other patients with what appeared to be a minimal tumour burden. Despite the disappointment that initial expectations have not quite been met, something extremely important has occurred in the treatment of cancer. For the first time, the controlled activation of a patient's endogenous immune system has been shown to have some promise as an antitumour treatment. Clearly, if the response rates reported to date are the best attainable its role will be limited. This remains doubtful as investigators are actively exploring combinations of IL-2 with other biologicals, cytokines, monoclonal antibodies and chemotherapeutic agents. Preclinical data suggest enhanced antitumour effects will be mediated by these combinations. While the magic bullet has not yet been found, there has been a major step forward since IL-2 was first described.

Previous work has utilized spleen colony formation to evaluate the fractional survival of AKR leukemia and normal bone marrow cells after in vitro heat exposure. An inherently greater sensitivity of neoplastic cells to thermal killing, as compared to normal syngeneic stem cells, has been established both at 41.8 degrees C and 42.5 degrees C. Normal bone marrow colony-forming units were assayed in lethally irradiated (750 cGy) mice. Leukemic colony-forming units were assayed in nonirradiated mice. Using this methodology, the authors demonstrated that the differential effect of hyperthermia on AKR murine leukemia and AKR bone marrow cells can be further enhanced by the addition of lidocaine or thiopental to incubation mixtures. These findings may have application to autologous bone marrow transplantation in humans.

The specificity of antigen recognition by in vitro sensitized human cytotoxic T lymphocytes (CTLs) has been studied using a sensitive cell-mediated lympholysis (CML) assay. Frequently, high levels of cytotoxicity are observed on third-party targets unrelated to sensitizing or responding cells; however, no cytotoxicity differing significantly from zero has been observed on targets autologous to the responding CTLs. This "cross-killing" of third-party target cells has been observed when stimulating and third-party cells bear no cross-reacting serologically defined (SD) antigens, thought to be the target antigens recognized by CTLs. CML-blocking studies, using unlabeled normal human lymphocytes to inhibit 51Cr release from radiolabeled target cells, have shown that cross-killing, even in the absence of shared SD determinants, results from CTLs recognizing antigens shared by the third-party targets and the initial stimulating population. Furthermore, these antigens have been mapped to the major histocompatibility complex (MHC). The ability of human CTLs to specifically recognize MHC-controlled antigens not detected serologically suggests that SD antigens may be recognized differently by alloantisera and CTLs, or that MHC antigens other than SD may be the targets of CTLs in CML.

The regulation of B-cell and T-cell immune responses has been extensively examined and in the experimental animal appears to involve regulatory or "suppressor" T cells (1-4). The limitations of in vitro experimentation have made comparable study of nonpathological human suppression quite difficult (5). We report here an in vitro method that generates and quantitates suppressor activity in man after antigen-specific activation in mixed leukocyte culture (MLC). The one-way MLC induces both a proliferative response (6) and the generation of cytotoxic T lymphocytes (CTLs) (7). Both of these responses are mediated by antigen-specific T-cell subpopulations (8,9) and have been correlated with recognitive and destructive phases of allograft rejection. Recent reports have examined the antigen reactivity of mouse (10,11), rat (12), or human (13,14) lymphocytes obtained after proliferation in MLC. In all cases, after the primary MLC proliferative peak, the recovered lymphocytes rapidly differentiate upon re-exposure to the initial stimulating population, but do so only weakly when exposed to a presumably noncross-reactive third-party stimulating population. Velocity sedimentation separation studies have shown that the blast cells produced in a primary MLC revert to small lymphocytes that rapidly differentiate into proliferating and/or cytotoxic T lymphocytes upon restimulation with the initial antigen (15). These findings demonstrate that positive selection for the responding population in primary MLC does exist and may account for at least part of the specificity of the secondary response. However, this positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection in not yet significant, suggesting that suppression may be overriding importance in the specificity of MLC-activated secondary responses.

Autologous marrow recovery without engraftment of donor marrow was observed after bone marrow transplantation (BMT) for two patients with acute lymphoblastic leukemia. Each had received marrow from a haploidentical mixed lymphocyte culture (MLC) reactive donor after pretransplant conditioning with total body irradiation and high-dose cyclophosphamide. To minimize graft-vs-host disease, the marrow was depleted of T cells in vitro by treatment with a monoclonal anti-T-cell antibody and complement. Two weeks after each transplant, reactive lymphocytes were noted transiently in the blood of each patient. Analysis of karyotype, HLA type, and in vitro MLC responsiveness proved the lymphocytes to be of host, not donor, origin. MLC studies showed rapid proliferative responses specifically to stimulating cells from the BMT donor, indicating in vivo sensitization to donor antigens. Return of hematopoietic function was markedly delayed, but it eventually normalized after several months, without evidence of chimerism. These studies confirm that some immune and hematopoietic stem cells of host origin survive the high-dose chemoradiotherapy used as transplant conditioning. Because these immune cells are specifically reactive to donor alloantigens, more potent suppression of host immunity may be needed to prevent nonengraftment of T-cell-depleted, HLA-mismatched bone marrow.

The mixed leukocyte culture (MLC) and the cell mediated lympholysis (CML) assays are used as in vitro models of the afferent, or recognitive, and efferent, or destructive, phases of the homograft reaction. Activity in both of these tests has been related to differences at the major histocompatibility complex, HL-A in man and H-2 in mouse. Recent evidence suggests that the presumed cell surface differences which lead to cell proliferation in MLC are different from those which act as a target for CML. Data are presented providing further support for this hypothesis; in addition separate cell populations may respond to the differences which activate cells in MLC and to the differences which serve as targets for CML. There thus appears to be a dichotomy both for genetic control of, and cell populations involved in, the recognitive and destructive phases of cell mediated immunity.