Assays Of Cell-mediated Immunity Research Articles

Influence of Mammary Tumor Progression on Phenotype and Function of Spleen and In Situ Lymphocytes in Mice<xref ref-type="fn" rid="FN2">2</xref>

Peripheral immunity to an immunogenic chemically induced mouse mammary adenocarcinoma has been demonstrated in the syngeneic host, i.e., BALB/c mice, by several in vivo and in vitro cell-mediated immune assays. Analysis of the responsiveness of the immune cells within the in situ population may prove to be more indicative of the actual interactions between host and tumors. A centrifugal elutriation procedure was used to isolate tumor-infiltrating lymphocytes. A fluorescence-activated cell sorter revealed that most in situ cells were of the T-cell lineage as determined by the absence of surface immunoglobulin (sIg) and the presence of the Thy 1.2 antigen on their surfaces, while in mice with large tumors there were 27.4% Thy 1.2+ and 47.9% sIg+ cells Further studies using fluorescein-conjugated monoclonal anti-Lyt 1 or anti-Lyt 2 antibodies revealed a decrease in the levels of Lyt 1+ cells and an increase in the percentage of Lyt 2+ lymphocytes in the spleens of mice bearing large tumors. Within the Thy 1.2+ population infiltrating mammary tumors, the proportion of lymphocytes presenting the Lyt 1 marker was decreased in comparison to that of spleens of normal mice (0.58 vs. 0.83). At the same time the relative proportions of Lyt 2+ cells within the Thy 1.2+ population was increased in the in situ population (0.50) compared to those of spleens of normal mice (0.28). Examination of the functional abilities of the in situ lymphocytes (ISL) revealed that ISL derived from small tumors responded to the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A), although at lower levels than those of the splenocytes of the animals from which they were obtained. Responses to PHA were lost rapidly in ISL from large tumors, whereas Con A responses were more durable. In contrast to the spleen cells of tumor bearers, ISL failed to respond to tumor-associated antigens (TAA) at any stage of the disease. The changes in the subsets of T-cells in the spleens of tumor-bearing mice and in ISL may provide an explanation for the decreased activation of ISL by mitogens and for the lack of responsiveness to TAA observed in the splenocytes of mice with large tumors and in the ISL isolated from the mammary adenocarcinomas.

Cell-mediated immunity: Correlation of mixed-leucocyte-macrophage migration inhibition with delayed-type hypersensitivity after immunization and donor-specific transfer of cell migration inhibition by dialyzable leucocyte extract

Active and adoptive sensitization of rhesus monkeys ( Macacca mulatta) as well as the development of a novel sensitive in vitro cell migration inhibition assay for cell-mediated immunity (CMI) in this species are described. First, the correlation of mixed leucocyte-macrophage migration tests (LMMI) with the whole blood lymphocyte transformation (LT) and the delayed hypersensitivity skin test (DH) in immunized animals are shown. Second, these tests are used to demonstrate adoptive transfer of specific/nonspecific cellular immunity (CMI) with dialyzable leucocyte extract (DLE) from immunized donor to unimmunized recipient monkeys. Seventeen animals were immunized with keyhole limpet haemocyanin (KLH) or hepatitis B surface antigen (HBsAg) in Freund's complete adjuvant (FCA) or with FCA alone. Acquisition of antigen-specific cellmediated immunity was detected by all three tests within 5 weeks of immunization. Positive LMMI responses were associated with positive DH and LT. However, there was no correlation between the magnitude or time of development of the three responses. Therefore, the LMMI test, like the LT test, is an in vitro parameter of DH, but reflects the activity of different subpopulations of lymphocytes and is regulated by different mechanisms. In addition, 12 naive animals received DLE. Within 3 weeks, transfer of sensitivity was detected towards antigens to which the recipients had previously not been reactive but the donors had been. An enhancement of transformation response to phytohaemagglutinin was also seen. Thus, rhesus DLE contains both donor-specific transfer factor-like and nonspecific adjuvant-like activities. In DLE recipients, unlike immunized animals, LMMI responses were dissociated from DH or LT responses in that positive LMMI was mostly seen with negative DH or LT to antigens. Therefore, LMMI emerged as the most sensitive assay for detecting adoptive transfer of CMI by DLE in vivo, supporting the view that different mechanisms regulate LMMI, LT, and DH.

Delayed cutaneous hypersensitivity in normals: choice of antigens and comparison to in vitro assays of cell-mediated immunity

In 81 normal subjects, ages 19 to 100 yr (mean 52), we studied the prevalance of positive 48 hr skin reactions to six antigens: fluid tetanus toxoid, Candida albicans, SK SD , Trichophyton, PPD, and coccidioidin. Of these, C. albicans was most frequently reactive (92%); SK SD (51%) and tetanus (49%) were less so. Each of the remaining three antigens was reactive in less than 42% of the subjects. The minimum number of antigens required to detect delayed hypersensitivity in 100% of subjects was two: C. albicans and tetanus. We found no correlation between skin reactivity at 20 min, 6 hr, and 48 hr for most of the antigens studied, suggesting different mechanisms for reactions occurring at each time. In 60 of the subjects, lymphocyte stimulation index (LSI) with tetanus toxoid and monocyte chemotaxis (MC) assays were done. The natural log of the area of induration at 48 hr after tetanus skin testing (148) increased as a function of LSI (p < 0.005) and MC (p < 0.025) by multiple regression analysis. Skin testing was less sensitive than LSI as a test for cell-mediated immunity in our population. However, because of availability and correlation with LSI, delayed cutaneous hypersensitivity should be tested initially. For this purpose, tetanus toxoid appears to be a useful antigen when used in combination with C. albicans.

Purification of a peptide antigen from non-human primate spermatozoa which is recognized by antibodies in sera of sterile patients

Active and adoptive sensitization of rhesus monkeys (Macacca mulatta) as well as the development of a novel sensitive in vitro cell migration inhibition assay for cell-mediated immunity (CMI) in this species are described. First, the correlation of mixed leucocyte-macrophage migration tests (LMMI) with the whole blood lymphocyte transformation (LT) and the delayed hypersensitivity skin test (DH) in immunized animals are shown. Second, these tests are used to demonstrate adoptive transfer of specific/nonspecific cellular immunity (CMI) with dialyzable leucocyte extract (DLE) from immunized donor to unimmunized recipient monkeys. Seventeen animals were immunized with keyhole limpet haemocyanin (KLH) or hepatitis B surface antigen (HBsAg) in Freund's complete adjuvant (FCA) or with FCA alone. Acquisition of antigen-specific cellmediated immunity was detected by all three tests within 5 weeks of immunization. Positive LMMI responses were associated with positive DH and LT. However, there was no correlation between the magnitude or time of development of the three responses. Therefore, the LMMI test, like the LT test, is an in vitro parameter of DH, but reflects the activity of different subpopulations of lymphocytes and is regulated by different mechanisms. In addition, 12 naive animals received DLE. Within 3 weeks, transfer of sensitivity was detected towards antigens to which the recipients had previously not been reactive but the donors had been. An enhancement of transformation response to phytohaemagglutinin was also seen. Thus, rhesus DLE contains both donor-specific transfer factor-like and nonspecific adjuvant-like activities. In DLE recipients, unlike immunized animals, LMMI responses were dissociated from DH or LT responses in that positive LMMI was mostly seen with negative DH or LT to antigens. Therefore, LMMI emerged as the most sensitive assay for detecting adoptive transfer of CMI by DLE in vivo, supporting the view that different mechanisms regulate LMMI, LT, and DH.

Immunopathogenesis of chronic pyelonephritis

Immunopathologic responses to urinary Tamm-Horsfall protein in the development of chronic pyelonephritis were examined by four different approaches. First, in a rabbit model, tubulointerstitial nephritis developed in 64 of 102 rabbits injected intravenously with urine or rabbit Tamm-Horsfall protein as compared with only one of 17 rabbits in two control groups. Circulating cytotoxic lymphocytes plus immunoglobulin G (IgG) antibodies against Tamm-Horsfall protein were found in 51 percent of challenged (urine or Tamm-Horsfall protein) rabbits with tubulointerstitial nephritis as compared with only 8 percent of those without it (p < 0.001). Second, in a porcine model of reflux nephropathy, 16 of 21 pigs with pyelographic findings indicative of reflux had elevated serum liters of antiTamm-Horsfall protein antibody as compared with 0 of 13 with normal pyelograms. Five of 10 refluxing pigs tested also had circulating lymphocytes that were cytotoxic in the presence of Tamm-Horsfall protein as compared with 0 of 13 with normal pyelograms. Third, in human studies, 12 of 49 patients with recurrent nephrolithiasis demonstrated abnormal elevations in anti-Tamm-Horsfall protein antibody; 13 of 49 had an abnormality in one of two assays of cell-mediated immunity to Tamm-Horsfall protein as compared with 0 of the normal control subjects. These abnormalities were not associated with overt obstruction or bacteriuria, but appeared to be more common in patients with recent onset and active recurrent nephrolithiasis. Lastly, an inhibitor of the binding reaction between human Tamm-Horsfall protein and its IgG antibody was detected in extracts of three uropathic coliforms. The inhibitors were partially purified by chromatographic means. Preliminary immunoautoradiographic studies revealed three or less protein-containing subunits of Escherichia coli that cross-reacted with anti-Tamm-Horsfall protein antibody. These studies suggest that autoimmune responses to Tamm-Horsfall protein may occur after exposure to Tamm-Horsfall protein by intravenous challenge, urinary reflux, or recurrent nephrolithiasis. This autoimmune response to Tamm-Horsfall protein may be the pathogenetic mechanism by which these factors, including bacteriuria, contribute to chronic pyelonephritis.

Correlation of in vivo and in vitro expression of cell-mediated immunity to mumps skin test antigen

The simplest,functional assay of cell mediated immunity, skin testing, is not always possible to do in every patient. A variety of in vitro assays, including lymphocyte transformation (LT) have been developed as possible alternatives to skin testing. In this report studies were undertaken to determine whether LT induced by mumps skin test antigen (MSTA) correlated with skin test sensitivity. LT stimulated by MSTA was performed with cells obtained from 4.3 normal subjects. Each subject was subsequently skin-tested with up to four concentrations of AISTA. LT results correlated well with skin test reactions. Lymphocytes from all subjects were responsive in LT assays. A less pronounced correlation was observed between the concentration of MSTA used for skin testing and LT. The results of these experiments suggest that LT assays with MS TA provide an adequate substitute for MSTA skin tests.

Impaired cell-mediated immunity in focal glomerular sclerosis.

Cell-mediated immunity (CMI) was evaluated in 8 patients with focal glomerular sclerosis (FGS), 50 patients suffering from chronic mesangial proliferative glomerulonephritis without renal insufficiency and 24 healthy controls. The following parameters were measured: delayed skin reactivity to purified protein derivative, circulating lymphocytes, lymphocyte cell-surface markers (neuraminidase-treated sheep erythrocyte and erythrocyte-antibody-complement rosettes) and functional markers (mitogenic responses to concanavalin A and phytohemagglutinin). The FGS patients with nephrotic syndrome (NS) had a significant depression in CMI, characterized by decreased responses of the lymphocytes to both concanavalin A and phytohemagglutinin, impaired delayed hypersensitivity to purified protein derivative and a decreased proportion of T lymphocytes as compared with normal subjects. In contrast, the levels of all CMI parameters studied in FGS patients in remission and in patients with chronic glomerulonephritis with or without NS did not differ from normal subjects. Thus, the majority of FGS patients with NS demonstrated an impaired response in a CMI assay system. The possible significance of these phenomena in the pathophysiology of FGS is discussed.

A program for the control of an image analysis system used in in vitro assays of cell-mediated immunity

A program for automated control of cell enumeration, microscope stage and focus adjustment and statistical analysis of data is described. The program is designed to run on an Hewlett-Packard 9835A desktop computer programmable in Hewlett-Packard Enhanced BASIC.

Cell-mediated immunity and its application in toxicology.

A variety of in vivo and more recently in vitro assays have been described to assess cell mediated immunity (CMI). Two methods routinely employed in our laboratory to assess CMI following exposure to chemicals in rodents include delayed hypersensitivity and in vitro lymphoproliferation. Preliminary studies indicate that depressed delayed hypersensitivity responses, as performed by a radiometric assay, correlates with altered susceptibility to infectious agents and tumor cell challenge following exposure to immunotoxic chemicals. Furthermore, suppression of T-cell lymphoproliferative responses to at least 50% below control values correlated with depressed delayed hypersensitivity responses and altered host susceptibility. On the other hand, when suppression of T-cell lymphoproliferative responses are within 50% of control values, delayed hypersensitivity and host susceptibility parameters are not affected. Assuming adequate technical expertise and accurate data interpretation, CMI assays of these types can provide a valuable data base for toxicology studies and immunotoxicity assessment.

Cell-Mediated Immunity and Its Application in Toxicology

A variety of in vivo and more recently in vitro assays have been described to assess cell mediated immunity (CMI). Two methods routinely employed in our laboratory to assess CMI following exposure to chemicals in rodents include delayed hypersensitivity and in vitro lymphoproliferation. Preliminary studies indicate that depressed delayed hypersensitivity responses, as performed by a radiometric assay, correlates with altered susceptibility to infectious agents and tumor cell challenge following exposure to immunotoxic chemicals. Furthermore, suppression of T-cell lymphoproliferative responses to at least 50% below control values correlated with depressed delayed hypersensitivity responses and altered host susceptibility. On the other hand, when suppression of T-cell lymphoproliferative responses are within 50% of control values, delayed hypersensitivity and host susceptibility parameters are not affected. Assuming adequate technical expertise and accurate data interpretation, CMI assays of these types can provide a valuable data base for toxicology studies and immunotoxicity assessment.

Detection of type-specific antigenic determinants of murine mammary tumor viruses with cell-mediated immune assays.

Exposure of lymphoid cells from normal and mammary tumor-bearing Balb/cCrgl mice to MMTV antigens result in positive responses in blastogenic transformation and migration inhibition reactions. The levels of these responses appear to depend on the murine strain source of the viral preparations. Enhanced reactivities were observed in cultures exposed to purified MMTV from culture fluids of mammary tumor cells of spontaneous origin in the C3H strain when compared to purified MMTV derived from milk of mice from the RIII strain. To ensure the virus relatedness of the differences observed, MMTVs obtained from a common source, i.e., the Crandall feline embryo kidney cell line, were employed in blastogenesis assays. Parallel testing of feline cell-derived C3H, RIII, and GR mammary tumor viruses resulted in different levels of [3H]thymidine incorporation by spleen cells from Balb/cCrgl mice over a range of viral concentrations. These results indicate that it is possible to detect various types of MMTV variants with cell-mediated immune assays. The diverse functional reactivities elicited by these viruses may be related to the differences observed in their bioactivities as determined by incidence and development of mammary tumors and the potential of causing metastases.

Kinetics of Responses to Tumor Antigens and Mouse Mammary Tumor Virus in BALB/cCrgl and BALB/cfC3H Mice&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;, &lt;xref ref-type="fn" rid="fn3"&gt;3&lt;/xref&gt;

Spleen cells from BALB/cCrgl mice responded to murine mammary tumor virus (MuMTV) in cell-mediated immune assays at higher levels than did the spleen cells from the syngeneic BALB/cfC3H mice. Implantation in BALB/cCrgl with a chemically induced mammary tumor and in BALB/cfCrgl mice with spontaneous mammary tumors (SMT) arising in the same strain resulted in sensitization of these animals to the antigens of their tumors. Reactivities peaked 3 weeks after transplantation, whereas no positive reactions could be detected when tumors reached maximum size. A kinetic study with the use of MuMTV antigen(s) showed that the responses of lymphocytes from BALB/cfC3H with SMT followed the same pattern as that obtained with tumor antigens, which indicated that this might be a de novo sensitization. In sharp contrast, a steady type of response to MuMTV was observed with the spleen cells from BALB/cCrgl mice; i.e., the levels of responsiveness to MuMTV did not significantly vary at any stage of tumor development. In vivo studies explored the possible relevance of the in vitro cell-mediated immunity to the host defenses. MuMTV-expressing mammary tumor cells were implanted in BALB/cCrgl and BALB/cfC3H mice. The total incidence of tumors was significantly reduced and a delay occurred in their time of appearance in BALB/cCrgl mice in relation to BALB/cfC3H animals. Thus the in vitro reactivity to MuMTV antigen(s) in BALB/cCrgl mice was found to be coincidental with a degree of protection against the development of MuMTV-expressing mammary tumors.

Cell-mediated immune assay in children with Schistosoma haematobium infection and the effect of niridazole therapy

Forty-five children with Schistosoma haematobium infections were studied utilizing a whole-blood culture technique to assay lymphocyte blast transformation responses. The mitogenic lectins, phytohaemagglutinin (PHA) and pokeweed mitogen (PWM), and heterologous schistosomal antigens from adult worms and eggs were used. Responsiveness to PHA was intact but PWM responses were significantly impaired. Varying responses to schistosomal adult worm antigens were evident. Responses to soluble egg antigens were consistently low. No correlation of lymphocyte transformation responses was evident with egg excretion rates or clinical data. Treatment, primarily with niridazole, resulted in augmented cellular immune responses to PHA, PWM and adult worm antigens two and eight weeks post-therapy. Anti-egg antigen responses did not significantly change. It was evident that niridazole did not induce cellular immunodepression. The role of schistosome-specific immune modulation is discussed.

Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats.

KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12-15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay (51Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing host.

Impaired cell-mediated immunity in lipoid nephrosis.

Cell-mediated immunity (CMI) was evaluated in 26 patients with lipoid nephrosis (LN), 50 patients suffering from chronic diffuse proliferative glomerulonephritis (CGN) without renal sufficiency and 24 healthy controls. The following parameters were measured: delayed hypersensitivity skin test responses to purified protein derivative (PPD) and candida, circulating lymphocytes. T lymphocytes and T lymphocytes with receptors for the Fc portion of IgG (T gamma cells) or IgM (Tmu cells). Patients with LN in relapse had less mean induration of skin reactivity and a smaller proportion reacting to both antigens as compared with the control subjects. In contrast, the intensity of skin reactivity and the frequency of negative reactions in patients with LN in remission and CGN were similar to those of the control subjects. It was also found that the LN patients in relapse had a significant T lymphocytopenia as well as a significant decrease in absolute numbers of Tmu and T gamma cells, whereas the patients with LN in remission and CGN did not differ significantly from the control population. Thus, the majority of patients with LN in relapse demonstrated an impaired response in a CMI assay system. The disturbed CMI may be secondary to hypoproteinemia and other nutritional factors induced by the nephrotic syndrome.

Cell-mediated immunity in idiopathic membranous nephropathy.

Cell-mediated immunity (CMI) was evaluated in 11 patients with idiopathic membranous nephropathy (MN), 50 patients suffering from chronic proliferative glomerulonephritis (CGN) without renal insufficiency and 24 healthy controls. The following parameters were measured: delayed skin reactivity to purified protein derivative (PPD), circulating lymphocytes, lymphocyte cell-surface markers (En and EAC rosettes) and functional markers (mitogenic responses to Con A and PHA). The MN patients with nephrotic syndrome (NS) had less mean induration of skin reactivity and a smaller proportion reacting to the PPD antigen as compared with the control subjects. In contrast, the intensity of skin reactivity and the frequency of negative reactions in MN patients in remission and CGN were similar to those of the control subjects. During the nephrotic stage of MN the proportion of T lymphocytes decreased with simultaneous increase of the proportion of B lymphocytes. It was also found that the MN patients with NS showed impaired lymphocyte reactivity with lower Con A and PHA responses compared to the normal controls. Conversely, the mean mitogenic responses to the antigens in patients with MN in remission and CGN were similar to those of the control subjects. Thus, the majority of MN patients with NS demonstrated an impaired response in a CMI assay system. The possible significance of these phenomena in the pathophysiology of MN is discussed.

In vitro assays of cell-mediated immunity in calves with persistent [formula omitted] infections do not correlate with delayed skin hypersensitivity

A long-term kinetic study of the immune response of four calves experimentally infected with a M. avium strain was made using the following tests: lymphocyte stimulation (LS), leukocyte migration inhibition (LMI), delayed skin hypersensitivity (DHS), and a radioimmunoassay (RIA) procedure for antibodies to M. avium . The cell-mediated immune (CMI) responses measured by these tests showed different courses during the infection period. The LS test showed several periods of peak values and the LMI test two peaks of responses of relatively shorter duration. At the end of the experimental period the DHS responses had decreased to insignificant levels, whereas the lymphocytes from all the calves responded in the LS test. No correlation could be detected between CMI and antibody mediated responses, and only two of the infected calves showed clear cut antibody responses measured by RIA. The results probably reflect the measurement of different aspects of the CMI response in the different test systems. The cyclical nature of the CMI measured by the LS and LMI tests may indicate the influence of regulatory mechanisms by suppressing lymphocyte sub-populations.

In Vitro Cell-Mediated Immunologic Assay for Cow's Milk Allergy

The production of a lymphokine, the leukocyte-migration-inhibition factor (LIF), by peripheral blood lymphocytes in response to an in vitro challenge with bovine beta-lactoglobulin was assayed in infants and children suspected of having allergy to cow's milk protein. Of the patients studied, 24 had cow's milk allergy, 24 were normal control subjects, 18 had recovered from milk allergy, 10 were newborns, and 10 were babies suffering from acute gastroenteritis. All patients with milk allergy demonstrated significant LIF production in response to beta-lactoglobulin (23.5% +/- 6.4%). In the normal control subjects, LIF was 3.1% +/- 4.3% (P < .0005). Only two of the 24 control subjects and two of the ten newborns had high-normal values bordering on the positive. None of the ten babies with acute gastroenteritis gave a positive response. Most of the children who had recovered from milk allergy and were ingesting cow's milk had negative assays. This cell-mediated immune assay is shown to be a reliable test for the diagnosis of sensitivity to milk protein in infants and children, and for determining dietary treatment and when this treatment can be safely terminated. In most cases, its use should eliminate the need for the potentially dangerous and ethically questionable provocation test, as well as the need for repeated intestinal biopsies.

Automation of the leukocyte adherence inhibition assay. Counting live mononuclear cells with an automated light microscope system

Adaptation of an automated light microscope system to the leukocyte adherence inhibition assay provides a rapid automated assay of cell mediated immunity. Comparison between the numbers of live mononuclear cells counted by eye and by machine yields no statistical differences in the per cent adherence or in the standard errors when performing either 10 or 20 replicate counts per antigen-cell mixture. Using the cell counter in a semi-automated mode, the counting is performed 10 times as fast in comparison to the manual method. A semi-automated system is described. The procedure requires from 4–6 × 10 5 mononuclear cells and from 2–3 μg crude KCl extract using a standard hemocytometer. The value of the technique lies in its availability as a rapid assay for both research applications and immunologic monitoring in the clinical laboratory.

Studies on the immunosuppressive properties of cyclosporin a in rats receiving renal allografts.

The immunosuppressive effects of cyclosporin A were tested in a DA (RT-1a) to Lewis (RT-1(1) rat renal allograft model, which represents a very strong histocompatibility barrier. Dose-response studies established that oral doses of 5 mg/kg/day or higher gave complete suppression of rejection, while oral doses of 2 mg/kg/day or lower were without effect. Intravenous administration of the drug approximately doubled its potency. Time studies showed that the period of administration was also critical, with a 7- or 14-day treatment course with 5 mg/kg/day orally giving prolonged graft survival, while a 4-day course was without effect. Large doses (up to 25 mg/kg/day orally) from day 4 after transplantation did not prolong graft survival, suggesting that cyclosporin A has no effect on an established rejection response. It was found that the lymphocytotoxin response to the graft was markedly suppressed by doses of cyclosporin A which maintained normal graft function, while lower doses had little or no effect on the lymphocytotoxin response. A cell-mediated immunity assay showed a substantial response, but one that was lower in amplitude from that of control animals. Histological study of 7th day allograft biopsies demonstrated essentially normal kidneys, except for a mild mononuclear cell infiltrate, at higher doses of cyclosporin. Lower doses of cyclosporin gave a picture of rejection no different from that seen in untreated controls. The LD50 of cyclosporin was found to lie between 50 and 100 mg/kg/day orally. Even the higher of these doses did not cause nephrotoxicity as determined biochemically and histologically.