Transfer factor from guinea pigs sensitive to dinitrochlorobenzene: absence of superantigen properties.

Transfer factor from guinea pigs sensitive to dinitrochlorobenzene was not bound to an immunoadsorbent column that is specific for the dinitrophenyl determinant. The absence of the dinitrophenyl determinant on transfer factor suggested that the factor does not function as superantigen. The duration of the adoptive sensitivity, the small molecular weight, and the polypeptide or polynucleotide (or a combination) composition of the transfer factor are consistent with a derepressor function of the molecule.

Immunity to tumor-associated antigens in vasectomized men.

Leukocytes from 49 vasectomized and 57 age-matched nonvasectomized men were tested in the leukocyte adherence inhibition (LAI) assay for reactivity to sperm and various 3-M KCl human tumor extracts. Forty-four percent of the vasectomized men and 15% of the control group were reactive to the sperm antigen preparation (P less than or equal to 0.03). Similarly, a significantly higher percentage of vasectomized men responded to 3 of 5 tumor extracts tested: melanoma I (34.7 vs. 15.8%, P less than or equal to 0.04), squamous cell carcinoma (48.8 vs. 26.0%, P less than or equal to 0.04), and breast carcinoma (19.5 vs. 4%, P less than or equal to 0.04). Thirty percent of vasectomized versus 4% of the control group responded to more than two tumor antigens (P less than or equal to 0.03). The degree of LAI reactivity to each tumor extract was highly correlated with degree of antisperm LAI reactivity, and the degree of LAI responsiveness to one of the melanoma extracts was significantly correlated with antisperm antibody titer as measured by the sperm-immobilization assay. Furthermore, nonresponsive leukocytes from the control population converted to tumor antigen-responsive when incubated with sera from vasectomized LaI-positive men. Data from this study indicated that a large percentage of vasectomized men with sperm immunity were responsive to tumor-associated antigens in the LAI test and that antisperm antibodies or other serum factors played a role in this response. These results and the evidence that most tumor antigen-responsive cancer patients also have serum antibodies that reacted with sperm suggested that vasectomized men and cancer patients frequently responded to immunologically cross-reactive antigenic determinants present on both sperm and tumor cells.

Immune response to melanoma extracts in three melanoma-prone families.

Immune reactivity to melanoma extracts was measured by the leukocyte adherence inhibition (LAI) test in 40 members of 3 melanoma-prone families. The melanoma patients had a wide range of responsiveness to the extract, the highest responder being a 10-year survivor. As a group, family members (including spouses) without disease had significantly elevated LAI responses compared to those of unrelated controls (P less than 0.01). Within the families, members with close exposure to melanoma patients for 10 years or more had a significantly higher response to melanoma antigen than did members with 0-5 years of close exposure (P less than 0.05). Responses of spouses and members at high risk of developing melanoma (B-K mole syndrome) also correlated with length of exposure to patients, which suggests that the elevated LAI response was not genetically determined. The high frequency of positive responses to melanoma antigens in these families, particularly in spouses, suggests the presence of transmissible melanoma-associated material capable of immunizing persons in contact with melanoma patients.

Flow cytofluorometric detection of tumor-specific rosette-forming cells in patients with squamous cell carcinoma of the head and neck.

Tumor-specific rosette-forming cells reactive to solubilized tumor antigens conjugated to autologous erythrocytes were quantitated by flow cytofluorometry. Leukocytes from a high frequency of the patients (greater than 70%) with squamous cell carcinoma of the head and neck (SQCC) formed rosettes to the conjugated SQCC tumor antigens but not to other histologically distinct tumor antigens (melanoma and colon carcinoma). Healthy control subjects or tumor patients with other cancers were mostly unreactive to the SQCC tumor extract [1 to 21 (5%) and 1 of 14 (7%) for controls and tumor patients, respectively]. Rosette-forming activity was observed in SQCC patients with primary cancers [22 of 30 (73%)] or in remission [5 of 6 (83%)], whereas patients with tumor recurrence were uniformly unresponsive [0 to 9 (0%)]. Tumor-specific rosette formation was mediated predominantly by monocytes, as identified by histochemical techniques and physiological properties. Rosette formation in reactive patients was abrogated by short-term culture, but the abated response could be restored by incubation with autologous serum or sera from other rosette-forming cell-positive patients. However, responsiveness of nonreactive patients with SQCC recurrence could not be constituted by rosette-forming cell-positive sera. These observations suggested the presence of tumor-reactive monocytes in a high frequency of patients with primary cancer or in remission but not in patients with recurrent disease.

Assessment of the mechanism of the leukocyte adherence inhibition test.

This study was designed to elucidate the mechanism of the leukocyte adherence inhibition (LAI) test in man. To identify the reactive cell types, enriched leukocyte populations (dextran-separated leukocytes and Hypaque-Ficoll-isolated mononuclear cells and neutrophils, as well as rosette-isolated B- and T-lymphocytes) were tested for leukocyte adherence in the absence of serum to tumor-specific antigens. LAI reactivity was not restricted to any of the enriched populations, suggesting the involvement of multiple cell types. Attempts to demonstrate soluble lymphocyte factors in the LAI mechanism have been uniformly negative. In contrast, factors in serum of immune donors were able to arm naive cells to be specifically responsive. This suggests a role for serum factors in the mechanism of LAI reactivity and partially explains the participation of multiple cell types in the responses observed. In additional studies, we could not document a correlation between the magnitude of the dermal test (delayed cutaneous hypersensitivity) and the magnitude of the LAI response in patients with squamous cell carcinoma of the head and neck. In 34 of 54 of these patients, there was agreement between the two tests (both positive, 27 of 54; both negative, 7 of 54). In the remaining 20 patients, the dermal test was greater than 5 mm while the LAI test was negative (less than 30% inhibition).

Association of melanoma tumor antigen activity with beta2-microglobulin.

The majority of melanoma tumor antigen activity present in melanoma extracts derived from fresh tumor tissue binds to a Sepharose-anti-beta2-microglobulin adsorbent. Removal of HLA antigens from the extracts of melanoma tissue by using a KBr flotation technique did not reduce either the tumor antigen activity of the extracts or the binding of melanoma tumor antigen (MTA) activity to the Sepharose-anti-beta2-microglobulin adsorbent. The complete blocking of MTA activity by pretreating the anti-beta2-microglobulin adsorbent with beta2-microglobulin and the lack of detectable MTA binding to a Sepharose anti-normal human serum adsorbent demonstrated the specificity of the binding of MTA to the anti-beta2-microglobulin adsorbent.

Automated counting of T cell rosettes.

Human T cell rosettes were enumerated using an automated particle counter, the Bio/Physics Cytograf 6300A. An electronic oscilloscope representation of particle absorbance and scatter of a focused laser beam allows the separation and enumeration of both rosetted and non-rosetted lymphocytes. Repeated Cytograf sampling of a single rosette preparation gave highly reproducible results, and sampling from replicate tubes produced the same degree of variation as microscopic analysis. T cell rosettes prepared from 27 volunteers and compared by both methods of quantitation showed a high degree of correlation. This method can objectively measure at least 100 times as many cells for their rosette-forming capability as the tedious microscopic technique.

Detection of antigen specific rosette formation with the FACS.

Donors previously sensitized to conventional antigens PPD and KLH were evaluated for their antigen binding responses, utilizing a rosette forming technique with antigen-conjugated autologous erythrocytes. Reactivity is directly correlated with prior sensitization. Furthermore, antigen specificity is suggested by inhibition of rosette formation by prior incubation with the relevant antigen. The frequency of RFCs detected cytofluorometrically was compared with conventional fluorescent microscopy determinations. RFCs detected in this manner were identified as antibody armed monocytes by cell depletion and histochemical studies. The usefulness of the rosette forming technique for the routine evaluation of donor immunity is discussed.

Delayed type hypersensitivity to gangliosides in the Lewis rat.

Systematic study of the immunologic properties of gangliosides has been hampered by the lack of a suitable assay. In this study, significant delayed type hypersensitivity reactions to gangliosides were observed in Lewis rats immunized with whole guinea pig spinal cord (GP-SC) in complete Freund's adjuvant (CFA). The reaction was manifested by an increase in ear thickness after intradermal injection of a mixture of gangliosides and methylated bovine serum albumin (mBSA). No responses were observed to either gangliosides or mBSA alone. The reaction to gangliosides increased after immunization, persisted for 48 h, and was characterized by perivascular infiltration of mononuclear cells. Further evidence for a cellular response was demonstrated by the transfer of ganglioside-specific ear swelling by cultured spleen cells. The response to gangliosides was not due to contamination with myelin basic protein (BP) since no reaction to gangliosides was observed in GP-BP/CFA-immunized rats, and no reaction to BP was observed in ganglioside/CFA-immunized rats. In BP-immunized rats, responsiveness to BP persisted after recovery from clinical EAE for at least 60 days. However, no response to gangliosides was observed in BP-immunized animals after recovery from clinical EAE, suggesting the lack of autosensitization to gangliosides due to the disease process itself.

Subcultured human endothelial cells can function independently as fully competent antigen-presenting cells.

Recent evidence has suggested that dendritic cells, epidermal Langerhan's cells and endothelial cells (EC) as well as macrophages, fulfill the requirements of antigen-presenting cells. Despite a variety of controls, one weakness in the evidence that these latter cell types can independently serve as antigen-presenting cells is that the cell preparations may contain small numbers of contaminating macrophages or other cell types. The experiments described in this paper are directed towards providing firm evidence that human EC are independently capable of presenting antigen to T cells. EC were isolated from human umbilical veins and maintained continuously by serial subculture for periods of up to 8 months. The subcultured EC displayed classic EC morphology and uniform immunofluorescent staining for Factor VIII-related antigen. The subcultured EC (tested to the 18th subculture) presented both particulate and soluble antigens to macrophage-depleted T cells with an efficiency equivalent to freshly isolated cells. Monoclonal antibodies to HLA-DR and HLA-DS determinants inhibited antigen presentation by either autologous macrophages or EC. In addition, antigen presentation by the subcultured EC was not affected by the macrophage-specific monoclonal antibody Mac-120, which inhibited antigen presentation by autologous macrophages in the same experiments. These results are consistent with human EC being able to independently function as fully competent antigen-presenting cells.

Endothelial cell presentation of antigen to human T cells.

Activation of human T cells requires presentation of antigen by Ia (HLA-DR in man) bearing cells of the mononuclear phagocytic series (macrophages, MO, and more recently Langerhans cells, dendritic cells, and vascular endothelial cells. Since T cells must cross endothelial barriers to enter extravascular tissues during immune reactions, we investigated the role of endothelial cells in antigen presentation. Endothelial cells were cultured from human umbilical veins and identified by classic morphology and specific markers (factor VIII related antigen, and so on). Antigen-pulsed endothelial cells were used to present antigen to MO-depleted human T cells; activation was assessed by 3H-thymidine uptake. The HLA-DR compatible endothelial cells were as effective as MO in reconstituting MO-depleted T-cell responses. The endothelial cell reconstituted responses were antigen specific, HLA-DR restricted, and blocked by monoclonal antibodies to HLA-DR framework structures. Moreover, the T-cell responses were clonal with respect to HLA-DR. A monoclonal antibody completely eliminated MO reconstitution of the MO-depleted response without diminution of endothelial cell reconstitution of the same response. Fibroblasts and smooth muscle cells cultured from the same umbilical veins could not reconstitute the MO-depleted T-cell response. These data indicate that endothelial cells play an important and distinctive role in lymphocyte triggering.

The mechanism of antigen presentation by endothelial cells.

Endothelial cells line the vessels and lymphatics forming a barrier between circulating T cells and the extravascular tissue site of antigen. We have suggested that circulating T cells recognize antigen on the surface of endothelial cells, resulting in the activation of the endothelium such that the endothelial cells then release the key mediators of a cell-mediated immune response. To test this hypothesis, we have evaluated the extent to which endothelial cells can signal antigen-specific T cell activation. We have shown that cultured endothelial cells are as effective as macrophages in lymphocyte activation and that this activation is HLA-DR restricted. In additional experiments we have established that endothelial cells synthesize both Ia and IL-1 early in the signaling process. To eliminate any possible contribution of other cell types participating in the T cell-endothelial cell interaction, we have shown that cloned endothelial cells present antigen to cloned T cells. Moreover, there appeared to be a preference of selected T-cell populations for different types of antigen presenting cells. These experiments document that endothelial cells are independently competent antigen presenting cells.

Influence of serum blocking factors on cancer patients undergoing immunotherapy.

Blocking factors are small polypeptide molecules that may appear in the serum of patients with cancer. These factors block the transformation of lymphocytes in culture to nonspecific mitogens such as phytohemagglutinin or concanavalin A and, therefore, may reflect changes in the immunocompetence of the patient. Blocking factors were monitored during the clinical course of thirty-five patients with cancer. These factors did not develop in patients with response to therapy whereas they did develop in patients without response. A third group of patients without response to therapy after a previous remission showed an absence of lymphocyte responsiveness in culture that was not due to blocking factors, suggesting that immune clone consumption had occurred. Dermal responsiveness to tumor antigen correlated with a favorable clinical course and was usually absent when serum blocking factors were present.

Transference of cell mediated immunity in patients with head and neck cancer.

A group of 67 patients with head and neck cancer has been studied of which 40 have received immunologic transfer factor from a normal donor pool. Examination of these patients revealed that lymphocyte reactivity to nonspecific mitogrens is depressed in patients who have head and neck cancer to a much greater extent than is seen in patients with other types of tumors. Furthermore, the depression is more prevalent among patients who have been treated with radiation. Patients in the head and neck group who have received transfer factor show an initial decreased response to PHA stimulation in culture. This is not seen in a control group of head and neck cancer patients or in patients with nonsquamous cander. Thymus-derived lymphocytes are depressed in patients with head and neck cancer, irrespective of whether they have received radiation. Th T-lymphocyte levels increased in eight of 38 patients who received nonimmune transfer factor, but 7 of these were in the group who had not received radiation. The leukocyte adherence inhibition (LAI) test has been used to determine tumor immunity in the patient test group. Changes in tumor immunity did not occur in those patients who received normal nonimmune transfer factor. Studies are presently in progress which provide for treatment of patients with head and neck cancer with specific squamous carcinoma immune transfer factor.

Assessment of immunocompetent cells in patients with head and neck squamous cell carcinoma.

Patients with squamous cell carcinoma of the head and neck have impaired T cell function and poor tumor-specific responsiveness. Disproportionate levels of circulating immunocompetent cells could be one reason for this diminished immunity. In this study, a panel of monoclonal antibodies and flow cytofluorometry were used to define the relative proportions of selected immune cell populations. We detected a deficiency of the interleukin-2-producing subset of T helper-inducer cells (TH 5.2+) in these patients. Our data showed no significant differences in circulating levels of total T cells, T cell subsets, B cells, monocytes, or natural killer cells when compared to age, alcohol- and tobacco-use matched controls.

Accessory cell function of the endothelial cell: its role in the cellular immune response.

We have proposed a hypothesis in which vascular endothelial cells, rather than or in addition to bone-marrow-derived cells, play an integral part in the antigen presentation event of cell-mediated immune phenomena including delayed-type hypersensitivity (DTH). Previously we have shown that a DTH ear-swelling response can be adoptively transferred in rats, using as few as 2 x 10(7) in vitro conditioned immune spleen cells. The transfer is antigen-specific, requiring the same sensitizing antigen in both the in vitro conditioning step and in the ear-test challenge. Adoptive transfer is also genetically restricted by alleles of the RT-1 region of the rat, requiring histocompatibility between immune donor cells and the naive recipient. In additional experiments, F1 to parental bone-marrow chimaeras were constructed such that the bone-marrow-derived cells and the non-bone-marrow-derived cells were of different RT-1 allotypes. When these chimaeras were used as adoptive transfer recipients, the transfer of DTH was possible only if the immune donor cells and the recipient non-bone-marrow-derived cells shared a common RT-1 haplotype, regardless of a shared haplotype with the bone-marrow-derived cells. These results point to a critical role for non-bone-marrow-derived cells (endothelial cells) in the DTH inflammatory response.