Search

A review of the literature on cell-mediated immunity to cancer revealed that many in vitro techniques have been used to investigate this subject. The techniques had been introduced to detect reactions between lymphocytes and tumour antigens, and serum factors which modify these reactions. Most of the methods were applicable to both animals and man and clinical uses in human cancer have been suggested, but the many disadvantages of existing methods seem to make them unsuitable for routine use.The leucocyte adherence inhibition (LAI) test was introduced as an improved technique in tumour immunology, and the original work of this thesis is concerned with the development, investigation and application of this techniqueAnti-tumour immunoreactivity to methylcholanthrene-induced tumours in mice was studied and specific cell-mediated immunity was demonstrated. Murine tumours showed individual tumour specificity. The LAI test was shown to depend on the production of a soluble factor which had characteristics in common with known lymphokines. It was produced by T-lymphocytes and its action was directed against adherent cells in the leucocyte-antigen mixtures. An indirect or 2-stage LAI test which depended on the presence of a soluble mediator was developed and this technique was used to study the kinetics of cell-mediated immunity in mice after tumour transplantation.Specific blocking of lymphocyte-antigen interaction was demonstrated in the serum of tumour-bearers and the kinetics of blocking in mice before and after tumour removal were investigated. Serum from mice after tumour removal was found to contain unblocking factors which interfered with the action of blocking factors in tumour-bearer serum.The LAI test was used to study immunoreactivity in human cancer. Four types of tumours were used: melanoma, colorectal carcinoma, medullary carcinoma of the thyroid and hepatoma. Patients were studied for reactivity to tumour antigens before and after tumour removal.The specificity of the LAI reaction with human tumours was related to the histological type of tumour rather than individual tumours. In hepatoma the situation was more complex and the LAI test was used to analyse the antigens involved. Serum blocking factors were detected consistently in patients with progressing tumours.The LAI test correlated well with the presence of tumour in both animals and man, and was shown to be a useful diagnostic aid in human cancer.

Human blood leukocytes from three subjects who had been contact sensitized to dinitrochlorobenzene were used in direct and indirect leukocyte-adherence-inhibition (LAI) reactions in an attempt to elucidate the cellular mechanism of reactivity. The leukocytes were separated and purified by standard procedures. In direct LAI, only T cells or populations containing T cells gave positive reactions (significantly reduced adherence) with the antigen. Supernatants from suitable leukocyte-antigen mixtures contained a soluble leukocyte-adherence-inhibition-factor (LAIF) that reduced the adherence of normal leukocytes. Only T cells or populations containing T cells were active in LAIF production; B cells, granulocytes, and monocytes were inactive. The cellular requirement for the action of preformed LAIF was not restricted: all major types of blood leukocytes were susceptible to its effect.

Blood leukocytes from patients with cancer (malignant melanoma; adenocarcinoma of colon, rectum, and breast) reacted with aqueous extracts of tumors of the corresponding type, with the result that adherence of the leu kocytes to glass was diminished. Leukocytes from normal individuals did not react. The leukocyte adherence inhibition (LAI) test could be completed in a few hours. Sera from tumor-bearing patients blocked the LAI reaction of their own leukocytes or leukocytes from other patients with the same type of tumor. Serum blocking activity was lost soon after surgical removal of the tumor; the patient’s serum then became unblocking. The LAI technique gave consistent results in a series of patients, analogous to those reported with the lymphocyte cytotoxicity test, and was easier and quicker.

Activated T cells synthesize and express a cell membrane-bound receptor for interleukin-2 (IL-2) and have recently been shown to secrete a soluble form of the same receptor. Hairy cell leukemia is a chronic disorder caused by expansion of a clonal population of an unusual mononuclear cell of B cell origin. These cells have previously been shown to express an IL-2 receptor on the cell membrane. The sera of 26 patients with hairy cell leukemia were examined for the presence of a soluble IL-2 receptor before and during therapy with either recombinant interferon alpha-2a or 2′-deoxycoformycin. Before therapy, all patients had markedly elevated levels of this soluble IL-2 receptor ranging from five to 60 times the highest level observed in normal control sera. In individual patients changes in the level during therapy correlated well with clinical assessments of tumor response; levels fell to near the normal range in patients responding to therapy. Patients not responding to interferon alpha had no significant change in the soluble IL-2 receptor level. These results suggest that hairy cells secrete a soluble IL-2 receptor and that serial measurements of the level of this receptor in the serum can be used as a noninvasive means to assess disease response to therapy.

Three patients with medullary carcinoma of the thyroid (MCT) were tested for antitumor cell-mediated immunity and serum blocking factors by the leukocyte adherence inhibition (LAI) test. Leukocyte reactivity with both autochthonous and allogeneic MCT extracts was detected after surgical removal of tumor. Blocking factors were present in patients with progressing MCT but disappeared after tumor removal; in one patient the serum then became unblocking. The leukocytes of seven control subjects did not react in the LAI test with MCT extracts. The value of the LAI test in MCT is discussed as an adjunct to the serum calcitonin assay.

Twenty-two normal volunteers had approximately eight, 2-hr-long leukapheresis procedures over a 2-year period and their natural killer (NK) cell function was prospectively measured. The NK activity of the preprocedure peripheral blood (pre-PB) was found to correlate well with the NK activity of the inital leukocytes removed by leukapheresis (I-Leuk). When the I-Leuk specimens were compared with the leukapheresis specimens removed at the termination of leukapheresis (T-Leuk), T-Leuk showed a consistent 10% increase in NK activity. When the pre-PB and the I-Leuk values were analyzed for each donor over the 2 years of the study, 18 donors revealed no significant change from their baseline NK activity, two donors showed a minimal increase in NK cell activity, and two donors displayed a minimal decrease in NK cell activity. We conclude that although leukapheresis appears acutely to boost NK cell activity, this increase is transient and small in magnitude. Most importantly, repeated leukapheresis does not appear adversely to effect this important effector function in normal donors.

The leucocyte adherence inhibition test provides a rapid, reliable, and specific technique for the immunodiagnosis of primary hepatocellular carcinoma (malignant hepatoma). The patient's blood leucocytes are tested in vitro for cell-mediated immunity against tumour-associated antigens and the serum is tested for blocking factor which interferes with the immunological reaction. Specific reactivity of both leucocytes and serum was consistently detected in patients with malignant hepatoma, and negative reactions were obtained in other liver diseases including secondary tumours of the liver. The test has provided positive evidence for the presence of hepatoma when more conventional methods gave doubtful or negative results. A positive result preceded the clinical appearance of tumour by up to three years in two patients.

Hairy cell leukemia is a lymphoproliferative disorder characterized clinically by cytopenias. Standard therapy following variable periods of disease stability consists of splenectomy that often restores normal hematologic parameters for periods ranging from weeks to years. Fifteen patients (five without prior splenectomy or chemotherapy) were treated with 3 X 10(6) units per day of recombinant leukocyte A interferon and 14 of 15 patients completed eight weeks of therapy and were evaluated for response. There was one complete and 12 partial responses for an overall response rate of 93 percent. All of these patients' conditions have remained in complete or partial remissions and they continue to receive interferon with a median follow-up of six months. Coincident with the normalization of peripheral blood counts was a return of natural killer activity and normalization of immunologic surface markers as determined by monoclonal antibodies. This study confirms and extends earlier observations with natural alpha-interferon and indicates that recombinant leukocyte A interferon in low daily doses is also very effective treatment for hairy cell leukemia. In fact, it may be the best single modality of therapy for inducing both hematologic and immunologic recovery of these patients and deserves consideration as initial therapy.

The ability of tumour extracts to augment E-rosette formation by cancer patients' lymphocytes was demonstrated for colorectal carcinoma, breast carcinoma and melanoma. Positive augmentation reactions were obtained with 14 to 20 patients tested with extracts related to their own tumour types. Some lack of specificity was suggested by 5 positive reactions in the same patients tested with unrelated extracts. No false positives were found in normal subjects. The technique is simple, rapid and appears to depend on tumour-associated antigens in the extracts. Simultaneous leucocyte adherence inhibition tests on split samples of blood had a high degree of sensitivity and specificity, confirming the potential reactivity of the leucocytes and extracts used.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN- alpha A). The binding of iodinated recombinant interferon-alpha to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN- alpha A. HCL patients (8/10) responded to rIFN-alpha A therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-alpha A therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN- alpha A therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

Lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) were administered by the ip route to patients with intra-abdominal malignancies. Pharmacokinetic studies of IL-2 revealed 10- to 100-fold higher concentrations of IL-2 in peritoneal fluid versus serum. Ip levels of IL-2 were maintained well above those required to generate and maintain LAK cells in vitro. LAK cell activity was detectable in the peritoneal fluid for the duration of each treatment cycle and did not disappear until IL-2 was discontinued. Detection of interferon-gamma (IFN-gamma) in the peritoneal fluid of all patients was consistent with production in situ by activated lymphocytes. In some patients, low but detectable levels of IFN-gamma were also found in the serum. In vivo activation of monocytes in the peritoneal fluid as measured by in vitro production of hydrogen peroxide was documented in the majority of patients. Neither interleukin-1 nor tumor necrosis factor-alpha was detected in the peritoneal fluid. We found no correlation between the presence or levels of IL-2, IFN-gamma, or LAK cell lytic activity in peritoneal fluid or serum and response or nonresponse to therapy.

The cytotoxic potential of mononuclear blood cells from 27 chronic schizophrenic patients was evaluated using quantitative measures of natural killer cell activity and macrophage inhibition of tumor cell growth. 44% of the patients had some evidence of deficient in vitro mononuclear cell function which was not correlated with a qualitative or quantitative alteration in the histologic appearance of these peripheral blood cells.

Eighty-one patients with a variety of refractory disseminated malignant neoplasms have been treated in the first multiple fixed-dose phase I trial of recombinant leukocyte A interferon (IFL-rA). Each patient received IFL-rA by intramuscular injection, three times weekly for 28 days. Dosages were escalated in different patients from 1 to 136×106units per injection. The toxic reactions seen with IFL-rA resembled those of nonrecombinant leukocyte interferon and included fever, chills, fatigue, anorexia, myalgia, headache, occasional nausea and vomiting, and dose-dependent reversible leukopenia and hepatic transaminase elevations. The pharmacokinetics of IFL-rA were also comparable with nonrecombinant leukocyte interferon. Objective evidence of antitumor activity was seen in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, breast cancer, and melanoma, indicating that IFL-rA, the first genetically engineered biological response modifier available for testing in cancer patients, is biologically active in vivo. (JAMA1982;248:2461-2466)

The susceptibility of human papillomavirus infection to polyclonal human leukocyte interferon (IFN-alpha) has been evaluated in patients with epidermodysplasia verruciformis (EV), a disease with extensive chronic papillomavirus-induced warts. In a double-blind, placebo-controlled study with intralesional IFN-alpha, four of five IFN-alpha-treated warts regressed; none of the placebo-treated warts responded (p = 0.024). Three patients with EV were treated with systemic IFN-alpha for 4 weeks in an open study, achieving partial regression of warts in all three. In a double-blind, placebo-controlled study, warts in two children with EV regressed with systemic IFN-alpha while two who received placebo showed no improvement. The lesions recurred following cessation of therapy. At the completion of therapy with IFN-alpha, histologic normalization was accompanied by a 95% decrease in the number of viral antigen-containing cells in the warts (p less than 0.001). We conclude that warts in EV respond to systemic and intralesional IFN-alpha.

PATIENTS RECEIVING freeze-dried skin (FDS) allografts were evaluated for cell-mediated response by a lymphocytotoxicity test. Eleven patients received single or multiple FDS allografts from a donor typed for human leukocyte antigen (HLA). Heparinized blood samples were drawn before the procedure and at 2, 4, and 8 weeks after grafting. Mononuclear cells were isolated by Ficoll-Hypaque gradients. Cell-mediated lymphocytotoxicity tests were conducted, with mononuclear cells from the FDS allograft recipients used as effector cells. Phytohemagglutinin-stimulated blast target cells of the same HLA type as that of the skin donor were labeled with chromium 51. Lympholysis was evaluated by measuring the amount of 51Cr release after 18 hours' incubation with target/effector cell ratios of 1:100, 1:50, and 1:25. Negative control wells contained target cells alone. Positive control wells contained target cells and effector cells sensitized in vitro against the target cells. Viability of effector cells was tested by trypan-blud dye exclusion and response to phytohemagglutinin. None of the experimental blood samples showed 51Cr release significantly greater than shown by the negative controls. Our findings of no cell-mediated response and the findings of a previous study showing no production of anti-HLA antibody in response to FDS allografts indicate that allogeneic FDS is an immunologically safe material for use in periodontal surgical procedures.

134 patients with a variety of malignancies were treated in two Phase 1 clinical trials of recombinant leukocyte A interferon (IFN) produced by recombinant DNA methodology by Hoffmann-La Roche Inc. in collaboration with Genentech Inc. IFN was given either twice daily or three times weekly for 28 days. Extensive monitoring of immune function was done on these patients, in an attempt to define the range of doses of interferon and timing of administration that would optimally alter immune functions which might have beneficial anti-tumor effects. These two protocols failed to result in an appreciable increase in NK activity but rather in the majority of patients there was no significant change in NK activity from the pretreatment baseline levels. A particularly unexpected finding was the depresssion in NK activity seen in 30% of the patients. In contrast, the IFN preparation did induce significant augmentation of monocyte function, as measured in a monocyte-mediated cytostatic assay, with increases observed in 80% of the patients. There is some evidence for the presence of a suppressor factor in the sera of these patients which interfered with in vitro boosting of NK activity by IFN and therefore might have been responsible for the paradoxical NK results. These data have also been analyzed in terms of their possible relationship to doses, and protocol of IFN administration, and to tumor response.

A total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-gamma), 6 patients by the i.m. and 5 patients by the i.v. route of administration. Dose ranges within each individual were from 0.05 mg/m2 of IFN (1 mg greater than or equal to 10 X 10(6) units of IFN) escalating to 10 mg/m2. All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chills, fatigue, anorexia, and granulocytopenia. The influenza-like symptoms were very similar to those encountered with IFN-alpha but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48-72 h following administration of rIFN-gamma. Absolute granulocyte counts only rarely dropped below 1000 mm3. Hepatotoxicity was not observed. IFN levels were determined by both a bioassay and an enzyme-linked immunosorbent assay. By the i.v. route, the peak level of IFN activity could usually be seen at completion of the infusion with a serum half-life of 30 min. By the i.m. route, the peak level of serum activity was generally detected between 4-8 h with a serum half-life of 4.5 h after the initial elimination phase. Peak IFN levels appeared to correlate with maximum toxicity. One patient with melanoma had a 25% reduction in a cutaneous lesion, but there were no other minimal, partial, or complete responses.

Interferon (IFN) has been shown to play a major role in the positive regulation of the activity of natural killer (NK) cells. All human IFN species tested to date of each of the three types, including homogeneous material and recombinant species, have had the ability to augment human NK activity. The mechanism for augmentation of this effector cell activity has been shown to be complex, with the potential for increasing the numbers of cells recognizing NK-susceptible targets, activating the lytic activity of target-binding cells, accelerating the rate of cytolysis of targets, and increasing the numbers of target cells killed by each effector cell. In addition, IFN has been shown to influence the growth and differentiation of NK cells from their progenitors: on the one hand, increasing the responsiveness of NK cells to interleukin-2 and thereby increasing cloning efficiency, and on the other hand, inhibiting generation of NK cells via a T cell-dependent mechanism. Some preliminary evidence has been obtained for the involvement of the 2′-5′A pathway in the mechanism of the augmentation of NK activity by IFN, since triphosphorylated 2′-5′A, after exposure of the cells to calcium chloride, caused an increase in NK activity. Nk cells appear to not only respond to IFN but also to produce IFN, in response to a wide range of stimuli. Overnight incubation of a highly purified population of human NK cells with viruses (herpes simplex or influenza), mycoplasma-contaminated tumor cell lines, BCG or C. parvum produced IFN-2, whereas under the same conditions, various mitogens (PHA, Con A, Staph enterotoxin) induced mainly IFN-α, and some IFN-β. Thus NK cells appear to be a major component of both the IFN-producing system and of the host effector mechanisms modulated by IFN. These observations have important implications in regard to clinical therapeutic trials with IFN or IFN inducers.

This study was undertaken to determine an immunologically active regimen for the administration of recombinant gamma-interferon (rIFN-gamma). The patient population included patients with completely resected melanoma, stage I (Clark's level IV or V) or stage II. All patients exhibited no evidence of disease (NED) at the time of the study. Patients received rIFN-gamma by intramuscular (IM) injection daily for 15 days at 0.0001 mg/m2, 0.001 mg/m2, 0.01 mg/m2, 0.1 mg/m2 (ten patients/group), or 0.25 mg/m2 (five patients). Interferon (IFN) was well tolerated, with non-dose-limiting constitutional symptoms occurring in the majority of patients at 0.1 mg/m2 and 0.25 mg/m2. All five patients receiving 0.25 mg/m2 developed elevated transaminase levels, which led to a discontinuation of therapy in one patient. Immunological activity was assessed by serial measurements of natural killer (NK) cell activity, hydrogen peroxide production by monocytes, and changes in expression of Fc receptors and human leukocyte class II antigen (HLA-DR) on monocytes. These changes were determined at baseline (X2), six to seven time points during rIFN-gamma therapy, and two times after the last dose of rIFN-gamma. No changes were observed at the two lowest doses. At the 0.01 mg/m2 dose, all parameters were elevated but not as consistently nor to the same levels as seen following administration of 0.1 mg/m2. At 0.25 mg/m2, H2O2 production was enhanced, but unlike at 0.1 mg/m2, it declined during the last few days of IFN therapy. Subcutaneous (SC) administration was compared with IM administration using the 0.1 mg/m2 dose. SC administration resulted in enhanced H2O2 production and Fc receptor expression by monocytes. More consistent elevations in peroxide generation and higher levels of Fc receptor expression were seen following SC administration. No significant difference was found between the two routes of administration. A comparison of two schedules, daily and three times weekly, suggested that monocyte activation may return to normal 72 hours after IFN administration. Of the doses tested, 0.1 mg/m2 administered daily appeared to be the most effective biological response modifier (BRM) regimen, and because of ease of administration, we favor the SC route.

A system has been developed for the isolation of large numbers of unfractionated mononuclear cells from single, well characterized normal individuals and for the separation by elutriation of these cells into populations of greater than 90% pure monocytes and greater than 99% pure lymphocytes. The total number of monocytes obtained from a single donor averaged about 550 million. After cryopreservation and thawing of these cells, the viability remained greater than 90%, 80% of original cells were recovered, and the ability to ingest antibody-coated targets was comparable to that of fresh monocytes. The cells remained sterile without the use of antibiotics and were suitable for long-term culture. The monocytes that were isolated and cryopreserved by these procedures functioned reproducibly as inhibitors of tumor cell growth and in an assay of responsiveness to monocyte migration inhibitory factor (MIF).

A two-stage modification of the leucocyte adherence inhibition (LAI) test is described, which quantitates cell-mediated immunity (CMI) in vitro with 0.5-ml samples of blood from mice exposed to methylcholanthrene-induced tumors. The total blood cells are washed and incubated for 30 min with tumor antigen, then centrifuged, and the supernatant assayed in the LAI test with normal peritoneal cells. The first stage of the reaction depends on the rapid formation of a soluble mediator which appears to be a lymphokine. By this method, the daily changes in specific CMI were followed in individual mice after syngeneic tumor transplantation.

Publisher Summary This chapter discusses modulation of natural killer (NK) activity by recombinant leukocyte interferon in advanced cancer patients. Interferons (IFN) are glycoproteins produced by certain cells after exposure to mitogens, viruses, or other agents. In vivo administration of IFN has also been reported to boost NK activity. Recently, it has been possible to clone the gene for human leukocyte A IFN, insert it into E. coli, and, thus, produce a recombinant leukocyte A IFN. A total of 108 patients were treated in two Phase I studies designed to evaluate the human tolerance, pharmacokinetic profiles and immunomodulatory effects of recombinant leukocyte A IFN. All assays were standardized for daily assay variation by the use of a set of three standards. The results of this study indicate that in vivo administration of IFN twice daily or three times weekly did not lead to an augmented NK response but instead to a diminished response in about 30% of the patients.

A variety of interferons have now been tested in clinical trials. Most of these trials have utilized leukocyte (α) interferon preparations. Early trials were conducted with partially purified material derived from the supernatants of virus-stimulated leukocytes that were of low purity and inconsistent pharmaceutical quality. More recently, trials have been conducted with a lymphoblastoid cell line interferon (α) of high purity and good reproducibility (Knost et al., 1983). Several recent studies have utilized recombinant α-interferon derived by cloning a gene for α-interferon in an E. coli expression system (Sherwin et al., 1982, 1983). Despite the major differences between these α-interferon preparations, many of the toxicities, immunological modulating effects, and therapeutic effects have been similar. Fever, chills, headache, fatigue, and anorexia have been rather constant side effects of these interferon preparations. At higher doses, mild hematological depression and transient hepatic enzyme abnormalities have been seen. Occasional cardiac effects including arrhythmias and ischemic effects have been observed in the context of these trials. Some central nervous toxicity including confusion, decreased ability to concentrate, and rarely seizures at very high doses have been seen in these studies. It is unclear whether all these effects are due to the direct action of the interferon preparation since the induction of fever, tachycardia, and fatigue may have secondary effects (Oldham, 1983a). The phase I trials for the α-interferons are virtually complete.

Further observations have been made using the in vitro leucocyte adherence inhibition (LAI) test previously described. The test has been slightly modified and shown to give highly reproducible results in detecting cell-mediated immunity in peritoneal cells of mice exposed to tumors, and in demonstrating factors which modify this immunity. Two different methylcholanthrene-induced tumors stimulated specific reactivity in their hosts as shown by the leucocyte adherence inhibition test employing soluble tumor extracts as antigens. When serum from tumor-bearing mice was added to the mixture, reactivity was specifically blocked. Serum from mice after tumor removal lacked the ability to block the leucocyte adherence inhibition reaction, but specifically reversed the effect of blocking serum (unblocking). Thus three important phenomena of tumor immunity, previously discovered by tedious and complex techniques, are now detectable by a brief, simple procedure.

The authors report the isolation of acquired immunodeficiency syndrome (AIDS) associated retroviruses from packed leukocytes of 2 chimpanzees inoculated intracerebrally and intravenously with brain tissue suspension from 2 patients with AIDS encephalopathy on days 7 and 14 after inoculation. Antibody to the AIDS-associated retroviruses has appeared in sera of 2 chimpanzees inoculated intravenously with plasma from different AIDS patients in 1 chimpanzee inoculated intravenously with brain and thymus suspension and in 1 chimpanzee inoculated intracerebrally with brain tissue suspension from a patient with AIDS encephalopathy. 11 chimpanzees inoculated with supernatant fluids from tissue cultures infected with human T-lymphotropic virus type III (HTLV-III) lymphadenopathy-associated virus (LAV) and IDAV have acquired antibodies to the LAV/HTLV-III viral antigens 2-8 weeks after inoculation. Virus was recovered from the lymphocytes of all 6 seroconverted animals between 8-154 days after primary inoculation of HTLV-III. 2 animals have severe suppression of T-cell function. However all 23 chimpanzees that have seroconverted to HTLV-III or LAV antigen have remained clinically well for 2-15 months of follow-up. There have been no tumors lymphadenopathy or severe opportunistuc infections. Other species of non-human primates similarly inoculated with HTLV-III and LAV have not seroconverted 2-10 months postinoculation. These findings confirm the active and persistent virus infection of chimpanzees with retroviruses derived from AIDS patients. They further establish the presence of viruses in the plasma and brain of AIDS patients by direct transmission of their virus to chimpanzees.