The immunogenicity of human HL-A haplotypesas measured by skin graft survival times and mixed leukocyte reactions.

The immunogenicity of the haplotypes in 30 families was measured by survival of skin grafts between selected paired family members. These families were genotyped for HL-A using 57 selected cytotoxic alloantisera defining HL-A 1-12 as well as other nondefined specificities. Mixed leukocyte reactions were also studied in this series and the correlations between the mixed leukocyte reactions with skin graft survival times, individual HL-A specificities, and the number of incompatible HL-A alleles are reported. Comments concerning the interpretation and quantitation of the mixed leukocyte reactions are discussed.

Renal transplantation between HL-A identical donor-recipient pairs. Functional and morphological evaluation.

16 patients underwent renal transplantation from a sibling donor who was prospectively determined to be ABO compatible and HL-A identical with the recipient. Unidirectional mixed leukocyte reactions were performed; in each instance, lymphocyte stimulation in either direction was not observed. The plasma creatinine 10-68 months after transplantation in these 16 patients ranged between 0.9 and 1.9 mg/100 ml. The creatinine clearance ranged from 48 to 113 ml/min, and the blood urea nitrogen (BUN) ranged between 12 and 35 mg/100 ml. Urine protein excretion varied from 0.11 to 1.86 g/day. Six patients exhibited no detectable clinical episodes of acute rejection; they were treated with azathioprine alone and each of them demonstrated normal or near normal renal histology when biopsy specimens were obtained more than 6 months after transplantation. Nine patients experienced acute rejection episodes that required the use of steroid therapy. The severity of these rejection episodes was variable; they included a mild reduction in renal function with an immediate steroid-induced restoration of function and eventual discontinuance of steroid therapy to severe reduction in function requiring prolonged and moderate doses of steroids without return to normal renal function. Renal histological observations in this group ranged from mild to marked cellular and structural changes which fit the criteria of the rejection. One patient demonstrated a gradual loss of renal function with heavy proteinuria. Biopsy of this allograft demonstrated the recurrence of original disease, i.e., lobular glomerulonephritis. The marked variability in the clinical course and allograft morphology in these 16 patients could be explained by antigenic differences at non-HL-A loci. The presence of minor histocompatibility loci has been well documented in other mammalian species and they are most certainly present in man. The need for their identification and definition is stressed.

Enzymatic susceptibility and spontaneous release of human melanoma tumor-associated antigens.

A chimpanzee anti-human melanoma antiserum was used to study the enzymatic susceptibility and spontaneous release into tissue culture medium of human melanoma tumor-associated antigens (TAA). Limited proteolytic digestion of melanoma cells with trypsin or with pronase rendered these cells refractory to lysis by the chimpanzee antiserum and complement. Longer periods of incubation of higher concentrations of enzyme caused an increased sensitivity to lysis. Digestion of melanoma cells with neuraminidase apparently exposed antigens reactive with natural antibodies in rabbit complement because cells so treated had a marked increase in sensitivity to cytolysis. Absorption of the complement with either neuraminidase-treated human melanoma cells or washed human spleen cells prior to its use in the cytotoxicity assay removed this activity. When absorbed complement was used, neuraminidase had no noticeable effect on the expression of malanoma TAA. These results suggest that proteolytic digestion of melanoma cells may prove to be a useful means of solubilizing TAA. The spontaneous release of melanoma cell membrane TAA was studied. Protein precipitated by (NH4)2SO4 from four of six samples of tissue culture medium used to feed malanoma cell lines contained significant antigenic activity compared to a control "antigen" preparation, whereas one preparation contained only limited TAA activity. One melanoma cell line that apparently failed to release TAA into the culture medium had previously become nonreactive with the chimpanzee antiserum. From these data, we conclude that melanoma cells growing in tissue culture rapidly release large amounts of TAA into the culture media and, as a result, the spent culture medium may be a good source for obtaining TAA for further study. The significance of these results is discussed.

Solubilization and partial isolation of human melanoma tumor-associated antigens.

Human melanoma cell membrane tumor-associated antigens (TAA's) were solubilized in an active form by pronase digestion of either a fresh melanoma or cells from a melanoma cell line maintained in tissue culture. Upon elution from Sephadex G-200 column, TAA's solubilized from the melanoma cell line were found in four distinct peaks that had apparent molecular weights of approximately 48,000 (partition coefficient Kd, 0.426), 25,000 (Kd, 0.567)8 17,000 (Kd, 0.699), and 13,000 (Kd, 0.831) daltons, respectively. Fetal antigen activity was found in all but the 13,000-dalton peak. HLA antigen activity was detected in the 17,000-dalton material. TAA's prepared from the fresh tumor source eluted from Sephadex G-200 column with an apparent molecular weight of 14,000-25,000 (Kd, 0.786-0.572) daltons, as did HLA antigens. A partial resolution of the TAA's from the HLA antigens was achieved with the use of DEAE-cellulose chromatography. Results of antigenic stability assays suggested that the TAA structure is stable to prolonged exposure to low pH. Recovery of TAA activity from the strong denaturing agents 5 m urea, 0.5% (wt/vol) sodium dodecyl sulfate, and 4 m guanidine hydrochloride was partially successful. These properties of the TAA's may be useful for further isolation of the TAA's.

Diagnosis of metastatic malignant melanoma by fine needle aspiration biopsy: a clinical and pathologic correlation of 298 cases.

Fine needle aspiration biopsies (FNABs) from 298 lesions in patients with suspected metastatic melanoma were studied. The results were correlated either with histopathologic diagnoses on resected lesions or with prolonged clinical follow-up. Of 165 malignant aspirates, 160 were confirmed either by surgical resection (65 cases) or by an appropriate clinical course (95 cases). Of the 107 benign lesions with adequate follow-up, 73 were confirmed as benign. There were 25 false negatives: 19 were inadequate samples, and 6 were presumed failures of needle localization. No interpretative errors were identified. Although 3 cases of FNAB-diagnosed malignant melanoma could not be confirmed by surgical biopsy, the cytologic findings were typical of malignant melanoma. Clinical follow-up, however, suggested that the cytologic diagnosis was in error. One case of a second unrelated malignancy (an adenocarcinoma of the lung) was correctly diagnosed with the use of FNAB. Because of its high degree of accuracy, FNAB has proved useful in the differential diagnosis of subcutaneous nodules, enlarged lymph nodes, and lung nodules.

Abnormalitieis of monocyte chemotaxis in patients with melanoma: effects of immunotherapy and tumor removal.

The chemotactic responsiveness of peripheral blood monocytes was studied before and after immunotherapy was administered to 56 patients with melanoma. Abnormal chemotaxis was found in 36 patients (64%) prior to treatment; this abnormality correlated with severity of disease and prognosis. Immunotherapy with BCG and sensitized autologous lymphocytes and X-irradiated melanoma cells or surgical removal of the neoplasm both reduced the percentage of patients with abnormal chemotactic responses. The best prognosis was found for those patients who had normal chemotaxis prior to therapy. The data support the hypothesis that abnormalities of monocyte function might render the host less likely to destroy developing neoplasms and that malignant tumors themselves might affect monocyte function.

Human cytotoxic T cells specific for autologous melanoma cells: successful generation from lymph node cells in seven consecutive cases.

Human T-cell populations specifically cytotoxic for autologous melanoma cells have been successfully generated from lymph node cells obtained from seven consecutive patients. The lymph node cells were stimulated in vitro with autologous irradiated melanoma cells; stimulation was repeated every 10-15 days at a tumor cell-to-lymphocyte ratio of approximately 1:20. Cytotoxic activity was assessed by a 4-hour 51Cr release assay. Mean lysis of autologous tumor cells was 47% at an effector-to-target cell ratio of 20:1, while mean lyses of the human myeloid leukemia cell line K562, allogeneic melanoma cells, and an osteosarcoma cell were 20%, 13%, and 11%, respectively. There was no lysis of autologous fibroblasts, fresh lymphocytes, or phytohemagglutinin-stimulated blasts. Three grades of specificity developed sequentially. In grade I, lysis of autologous tumor cells exceeded lysis of allogeneic tumor cells but did not exceed lysis of K562 cells. In grade II, lysis of autologous tumor cells exceeded lysis of K562 cells and all allogeneic tumor cells tested. In grade III, potent lysis of autologous tumor cells (greater than 40%) exceeded lysis of K562 cells and of all allogeneic tumor cells tested. All seven lymphocyte populations reached or exceeded grade I. Six reached or exceeded grade II. Two progressed to grade III. The generated cells were T cells, as determined by phenotypic analysis with flow cytometry. CD4+ cells and CD8+ cells accounted for 83%-100% of the cells. CD8+ T cells were separated from CD4+ T cells by panning with OKT8 and OKT4 antibodies. The resulting CD8-enriched and CD4-enriched populations were compared as effectors in cytotoxicity assays. The results suggest that the cell responsible for lysis of autologous tumor cells is CD8+. The methods used in this study have repeatedly resulted in the successful generation of cytotoxic T lymphocytes specifically cytotoxic for autologous melanoma cells; it is suggested that these cells have potential application for adoptive immunotherapy of melanoma.

Randomized trial of high-dose chemotherapy with autologous bone marrow support as adjuvant therapy for high-risk, multi-node-positive malignant melanoma.

BACKGROUND: Chemotherapy adjuvant to surgery in metastatic melanoma has been evaluated in only a few prospective randomized trials. In the treatment of metastatic melanoma, dacarbazine has response rates of 15%-25% and in several studies, when combined with other alkylating agents, has yielded even higher response rates. Among the highest response rates are those achieved by using high-dose chemotherapy regimens combined with autologous bone marrow support (transplantation). PURPOSE: We conducted a prospective randomized clinical trial to test the efficacy of high-dose alkylating agents in combination with autologous bone marrow support given as adjuvant therapy for high-risk stage II (World Health Organization) melanoma. METHODS: Thirty-nine patients with metastases involving five or more lymph nodes were randomly assigned to one of two treatment arms within 8 weeks of lymphadenectomy: immediate treatment or observation only. The immediate-treatment arm consisted of 19 patients who, immediately after random assignment, received high-dose chemotherapy with alkylating agents, followed 3 days later by reinfusion of autologous bone marrow. The observation arm consisted of 20 patients who were observed until relapse (confirmed by biopsy) and were then treated with the identical high-dose alkylating agent chemotherapy followed by reinfusion of autologous bone marrow. Bone marrow was harvested from the patients under general anesthesia 1-2 weeks prior to chemotherapy and was cryopreserved. Chemotherapy consisted of intravenous administration of cyclophosphamide (1875 mg/m2 as a 1-hour infusion daily for 3 days), cisplatin (55 mg/m2 per day by continuous infusion over the same 72-hour period), and carmustine (BCNU) (600 mg/m2) given immediately after cisplatin on the 4th day as a 2-hour infusion. The total doses of the three drugs were 5625, 165, and 600 mg/m2, respectively. All patients received medical evaluations every 6-12 weeks over the study period. Kaplan-Meier estimates were used to determine the time to disease progression on the basis of intent to treat. RESULTS: There was no statistically significant difference in overall survival or in time to disease progression between the two treatment arms. However, the median time to progression was 16 weeks in the observation arm and 35 weeks in the immediate-treatment arm. CONCLUSIONS: Immediate adjuvant high-dose chemotherapy with alkylating agents followed by autologous bone marrow support more than doubled the time to disease progression compared with observation alone, though the difference was not statistically significant. No differences in overall survival were noted.

Characterization of a chimpanzee anti-human melanoma antiserum.

An antiserum to human melanoma has been produced in a chimpanzee by hyperimmunization with melanoma cells from a single donor. After absorption with the peripheral blood lymphocytes of the tumor donor, this antiserum is specifically cytotoxic to melanoma cells from 14 tissue culture cell lines and to cells from 8 fetal fibroblast cell lines. Peripheral blood lymphocytes-absorbed antiserum is negative when tested against a large panel of normal peripheral blood lymphocytes and fibroblasts and against eight non-melanoma neoplastic tissue culture cell lines. Subsequent absorption with melanoma cells from any of seven sources removes all antimelonoma and all antifetal reactivity of the antiserum. Similar absorption with fetal cells removes all antifetal activity but does not completely remove the antimelanoma activity. The immunoglobulin G fraction of the antiserum is shown to possess the cytotoxic activity.

Generation of primary tumor-specific cytotoxic T lymphocytes from autologous and human lymphocyte antigen class I-matched allogeneic peripheral blood lymphocytes by B7 gene-modified melanoma cells.

Expression of B7.1 costimulatory molecules on tumor cells has been shown to elicit antitumor immunity in mice. In the present study, we have developed a human B7.1 retroviral vector system to effectively transduce human melanoma cell lines and investigated the potential role of B7.1 in the generation of tumor-specific CTLs from peripheral blood lymphocytes (PBLs) in vitro. We have demonstrated that B7.1-modified melanoma cells are able to induce primary CTL activity from autologous, human lymphocyte antigen (HLA) class I-matched allogeneic PBLs and purified CD8+ T cells in the absence of exogenous cytokines. CTLs generated by B7.1 are tumor specific and HLA class I restricted, and CD8+ T cells are primarily responsible for this specific cytotoxicity. Furthermore, CTLs generated from HLA class I-matched PBLs by B7.1 are cytolytic to tumor cells autologous to the stimulated PBLs. These data suggest that B7.1-modified tumor cells can be used as a potent tumor vaccine for both autologous and HLA class I-matched allogeneic patients.

Human xenograft-nude mouse model of adoptive immunotherapy with human melanoma-specific cytotoxic T-cells.

We investigated the efficacy of human melanoma-specific cytotoxic T-cells (CTLs) in treating experimental human melanoma metastases in a nude mouse model of adoptive immunotherapy. Hepatic metastases were generated by the intrasplenic injection of 1.5 x 10(6) human melanoma cells. Animals were then randomized to receive saline, interleukin-2 only, or CTLs and interleukin-2. CTLs were effective when administered 3 or 7 days after generation of hepatic metastases, with 96 and 88% of animals disease-free, respectively, when examined at one month. Interleukin-2 alone was not effective. In addition, CTLs were effective when as few as 2.5 x 10(6) T-cells were adoptively transferred. Only 33% of the animals were tumor-free when CTLs were administered on day 10, and CTLs were not effective when given at day 14. Human CTLs that were not cytotoxic for the tumor line used in vivo, when tested in a 51Cr assay, were also not effective in the model of immunotherapy. This suggests that the tumor-specific CTLs maintain their specificity in vivo, and eliminates a nonspecific inflammation directed against the human CTLs as a possible cause of the antitumor effect. These studies lay the foundation for clinical trials of CTLs in the adoptive immunotherapy of patients with metastatic melanoma.

Tumor membrane lymphocyte stimulation assay in patients with renal cell carcinoma.

Peripheral blood lymphocytes from 15 patients with hypernephroma were stimulated with partially purified tumor plasma membranes to incorporate [3H]thymidine. Kidney tumor and normal kidney membranes were adjusted to antigenic equivalence as determined by their ability to inhibit in the HLA 51Cr microcytotoxicity assay. Membranes from control "normal" kidney adjacent to the tumor stimulated less than did the tumor. Six of eight patients responded to autologous tumor (p less than 0.05). One patient responded to allogeneic tumor of the same histological type. The importance of statistical analyses of tumor membrane lymphocyte stimulation data is discussed in relation to the assay system. Sequential studies suggest that this assay may be useful as a guideline for the monitoring of current therapeutic regimens and future immunotherapy. The results of this assay are discussed in relation to other in vitro tumor lymphocyte stimulation assays. The limitations of this assay appear to be two: (a) it can be used only in large tumor systems where there is adequate tissue for analysis and controls; (b) it may detect nontumorous antigens or nonspecific stimulators in allogeneic studies. Further studies are needed to correlate the blastogenic response with the patient's prognosis.

Elevation of topoisomerase I messenger RNA, protein, and catalytic activity in human tumors: demonstration of tumor-type specificity and implications for cancer chemotherapy.

Topoisomerase I has been identified as an intracellular target of camptothecin, a plant alkaloid with anticancer activity. Various lines of evidence suggest that the sensitivity of cells to this drug is directly related to the topoisomerase I content. In humans, the levels of topoisomerase I have been shown to be elevated in colorectal tumors, compared to normal colon mucosa. The aim of our study was to determine whether (a) topoisomerase I levels are elevated in other solid tumors, (b) the elevated enzyme is catalytically active in these tumors, and (c) the increase in topoisomerase I levels in colorectal tumors is a result of increased transcription or translation. Topoisomerase I levels were quantitated in crude extracts from colorectal, prostate, and kidney tumors and their matched normal counterparts by Western blotting and by direct determination of catalytic activity, and mRNA levels were determined by Northern blotting. By Western blotting, colorectal tumors showed 5-35-fold increases in topoisomerase I levels, compared to their normal colon mucosa. In the case of prostate tumors, the increase was 2-10-fold, compared with benign hyperplastic prostate tissue from the same patients. However, no difference was observed in topoisomerase I levels in kidney tumors, compared to their normal counterparts. The catalytic activity of topoisomerase I was determined by a quantitative 32P-transfer assay in crude homogenates, without isolating nuclei. Colorectal and prostate tumors exhibited 11-40- and 4-26-fold increases, respectively, in catalytic activity. However, kidney tumors did not show any alteration in catalytic activity, compared to their normal matched samples. Thus, for all three tumor types there was a good correlation between enzyme levels and catalytic activity. Finally, colorectal tumors were analyzed for steady state mRNA levels. A 2-33-fold increase in mRNA levels was found in colorectal tumors, compared to normal colon mucosa. These results suggest that alterations in topoisomerase I expression in humans are tumor type specific and that the increase in topoisomerase I levels results from either increased transcription of the topoisomerase I gene or increased mRNA stability.

Generation of human autologous melanoma-specific cytotoxic T-cells using HLA-A2-matched allogeneic melanomas.

Autologous tumor-specific cytotoxic T-lymphocytes (CTLs), generated by repeated stimulation with autologous melanoma and expanded in interleukin 2, are major histocompatibility complex restricted. These CTLs recognize a common tumor-associated antigen in the presence of HLA class I determinants, suggesting that allogeneic melanomas which express the restricting HLA-A region antigen could substitute for the autologous tumor in the generation of CTLs. This was investigated in the HLA-A2 system. Four T-cell lines were established by stimulation of lymphocytes with either autologous tumor or an HLA-A2-matched allogeneic melanoma. Allogeneic stimulated CTLs specifically lysed the autologous tumor and demonstrated an identical pattern of HLA-A2 restriction, when compared to the autologous stimulated CTLs. Lysis by the allogeneic stimulated CTLs was blocked by a monoclonal antibody to HLA class I antigens; lysis was also inhibited by both autologous tumor or HLA-A2 allogeneic melanomas when evaluated in cold target competition studies. The allogeneic stimulated CTLs proliferated in response to both autologous tumor and HLA-A2 melanomas, but not in response to HLA-A2 nonmelanomas. By phenotypic analysis these CTLs were CD3+ and predominantly CD8+ cells. We conclude that autologous tumor-specific CTLs can be generated using HLA-A region-matched allogeneic melanomas for stimulation. Since established, HLA-typed melanoma tumor lines can be used in the absence of autologous tumor; this procedure can be applied clinically to a broad patient population and may prove useful in the adoptive immunotherapy of melanoma.

Eradication of melanoma pulmonary metastases by immunotherapy with tumor cells engineered to secrete interleukin-2 or gamma interferon.

This study was undertaken to investigate the effectiveness of interleukin-2 (IL-2) and gamma interferon (gammaIFN)-modified B16 melanoma cells in the immunotherapy of established melanoma pulmonary metastases. The genes for IL-2 and gammaIFN were introduced retrovirally into B16 melanoma cells. Transduction with the gammaIFN, but not the IL-2, gene caused significant increases in the expression of major histocompatibility complex (MHC) antigens on B16-gammaIFN cells. The in vivo tumor-forming capacity of both IL-2- and gammaIFN-transduced B16 cells was drastically reduced when the cells were inoculated subcutaneously (SC) in syngeneic C57BL/6 mice. After intravenous (IV) inoculation, most of the B16-gammaIFN cells were rejected, but B16-IL-2 cells were relatively tumorigenic and formed pulmonary metastases. C57BL/6 mice bearing 4-day established parental B16 lung metastases were treated with B16 parental (B16P) unmodified cells, IL-2- or gammaIFN-modified B16 cells, or a combination of both transduced cells. Treatment consisted of a weekly intraperitoneal (IP) injection of one million irradiated (10,000 rad) tumor cells alone or in combination with exogenous IL-2 for a total of three to four injections. Immunotherapy with B16 parental or B16-IL-2 secreting cells caused a moderate reduction in the number of lung metastases. However, mice treated with gammaIFN-secreting B16 cells showed a significant reduction or complete elimination of lung metastases. There was no additive effect for combining both IL-2- and gammaIFN-modified tumor cells in the immunotherapy. Exogenous IL-2 (50,000-100,000 U/day for 3 days) caused a significant enhancement of the immunotherapeutic benefit of the vaccines. Moreover, mice treated with gammaIFN-modified B16 cells survived longer than the other groups. Twenty-five percent of these mice were tumor free and remained alive for an observation period of 4 months. The in vitro cytolytic activity of splenocytes in chromium release assays did not correlate in every case with the in vivo antitumor effect of the treatment. Our findings have implications for the use of cytokine-modified cells for immunotherapy and for evaluating the therapeutic benefit of this novel treatment.

Transduction of human melanoma cells with the gamma interferon gene enhances cellular immunity.

Human tumor cells transduced with the gamma interferon (gamma IFN) gene are currently used in specific active immunotherapy protocols to enhance the antitumor immune responses of cancer patients. This in vitro study was undertaken to examine the initial events in the cellular immune response that may occur following the administration of the gamma IFN-transduced cell vaccine. Human melanoma tumor cell lines were transduced with a MoMLV-based retroviral vector carrying the human gamma IFN gene. The transduced cells expressed the cytokine gene, secreted biologically active gamma IFN, and exhibited enhanced expression of MHC class I and class II (HLA-DR), and ICAM-1 surface antigens. The gamma IFN-transduced and corresponding parental melanoma cells were used for the induction of short-term lymphocyte cultures. Peripheral blood lymphocytes or lymph node cells from 20 melanoma patients were stimulated for 5 to 15 days with autologous or MHC class I-matched allogeneic parental or gamma IFN-transduced melanoma cells. Seven of the 20 lymphocyte cultures showed substantial increases in lytic activity following stimulation with the transduced melanoma cells in comparison to control lymphocyte cultures stimulated with unmodified parental melanoma. The cytolytic activity stimulated with gamma IFN-modified melanomas was mediated partly by MHC-restricted cytotoxic T lymphocytes and partly by NK cells. Lymphocyte cultures that displayed increases in cytotoxicity after stimulation with the gamma IFN-transduced melanoma cells also exhibited enhanced expression or induction of one or more of the following lymphokines: IL-4, IL-1 alpha, IL-1 beta, gamma IFN, and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of non-human primates for production of antisera to human tumor-associated antigens.

High-titered antisera against human melanoma- and leukemia-associated antigens have been produced in non-human primates. The data from this and from other studies suggest that this model may prove a valuable source of highly specific antisera against a variety of human tumor-associated antigens or alloantigens. Our methods for production and characterization of these antisera are reported.

Membranoproliferative glomerulonephritis. Progression from the pure form to the crescentic form with recurrence after transplantation.

Described are the clinical course and renal morphologic findings in a patient with membranoproliferative glomerulonephritis. intially, the patient had a pure form of membranoproliferative glomerulonephritis but after a 3 year course, it became crescentic. After a renal allograft was performed, membranoproliferative glomerulonephritis recurred within 1 month in a pure form and subsequently developed into the crescentic form. This change occurred in the host kidney as well as in the allograft immediately after immunosuppressive and steroid therapy was discontinued.

Immunological consequences of transplanting rat insulinomas.

We investigated the genetics of transplanting a NEDH rat insulinoma in various donor-recipient combinations. The insulinoma survived indefinitely when it was transplanted in the NEDH strain. Mean survival times of 8.6 +/- 1.2 and 3.3 +/- 1.0 days were recorded in allogeneic and xenogeneic combinations, respectively. No prolongation of mean survival time was found when the rat insulinoma was first passed through the athymic nude mouse and then transplanted as either an allograft or xenograft. The findings of the major histocompatibility antigen (Ag-B7) on the original insulinoma and the tumor that had been passed through the nude mouse demonstrates no demonstrable loss of transplantation antigens during passage.

Factors which have a significant effect on the survival of human skin grafts.

Survival of 436 ABO-compatible skin grafts exchanged in 97 Caucasian families was prolonged if donor and recipient were genotypically, as compared with phenotypically, HLA identical. Among skin grafts between haploidentical family members, a mismatch at the A locus was equivalent to a mismatch at the B locus. Skin grafted from child to mother survived longer than did skin grafted between other family members, other variables being equivalent. A highly significant positive correlation was found between the age of recipient and skin graft survival. In addition, a significant interaction was found between the relationship of donor and recipient and degree of antigen match.