AN APPROACH TO THE QUANTITATION OF IMMUNOGENIC ANTIGEN

Using passively administered isotope-labeled anti-KLH to suppress the antibody response of rabbits to KLH, we have attempted to estimate the amount of antigen actually involved in stimulating antibody formation. Single and paired label tracer studies of passively administered anti-KLH IgG indicated that from 0.7 to 2.9 µg were utilized or involved by the antigen in the course of a 90% suppression of the response to 2 mg KLH. Tracer studies of labeled anti-KLH F(ab')2 fragments revealed the retention of from 2 to 3 µg of these fragments in the entire rabbit during a 60% suppression of the response to 1 mg KLH. Based on previously determined ratios of mixtures of KLH and suppressive amounts of anti-KLH in adjuvant, the antibody utilization data were converted to the probable amount of antigen or immunogen involved. It appears that after an injection of 2 mg of KLH approximately 0.2–0.5 µg of antigen persisted and reacted with antibody given 24 hr later. Since all of this persisting, reactive antigen may not be immunogenic, the above estimate of immunogen is probably high, but may serve to establish upper limits for the amounts of immunogen involved in stimulating antibody formation and provide a meaningful frame of reference for antigen tracer studies

SEPARATION OF STAGES IN THE DEVELOPMENT OF THE "T" CELLS INVOLVED IN CELL-MEDIATED IMMUNITY

Density distribution analysis in continuous gradients of albumin has been used to study the development of cytotoxic lymphocytes (CL), to separate and characterize the progenitors of CL, and to determine their relationship to subpopulations of T cells. CL progenitors in the thymus were a homogeneous, medium-density population, distinct from the typical, dense, thymus small-lymphocyte. Activity seemed to be associated with one minor subpopulation of cells with surface antigenic properties characteristic of peripheral T cells (high levels of H-2 antigen, low levels of θ-antigen). CL progenitors in the spleen differed from those in the thymus and normally had the high buoyant density of typical small T lymphocytes. In states of antigenic stimulation, some lighter-density CL progenitors were found in the spleen. The buoyant density of the CL population developing in the spleens of immunized animals showed progressive changes with time. Early, "immature" CL had the light-density characteristics of large, activated lymphocytes. As the response developed, the density of the CL population increased, and finally approached that of CL progenitors and of typical small lymphocytes. The data suggest that density subpopulations of T cells represent stages in the development of immunocompetent cells. Possible differentiation pathways of T lymphocytes in the thymus and in the spleen are discussed

COMPARISON OF THE IMMUNE RESPONSIVENESS OF NZB AND NZB x NZW F1 HYBRID MICE WITH THAT OF OTHER STRAINS OF MICE

The immune responsiveness of (NZB x NZW) F1 hybrid mice (NZB/W) has been compared with that of three other strains of mice, A/J, BALB/c, and CBA/J. The antigens used included sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and human γ-globulin (HGG). It was found that important strain differences existed in the amount of antibody produced, but the relative immune responsiveness depended very much upon the nature of antigen. By comparison with the other strains tested, NZB/W mice had a higher antibody production to some antigens (SRBC and BSA) but were low responders to others (KLH). Induction of unresponsiveness to HGG by treatment with ultracentrifuged HGG was studied in the strains cited above. NZB/W mice became tolerant after injection of HGG ultracentrifuged at 100,000 g for 2 hr. Similar experiments carried out with another preparation of HGG (centrifuged at 20,000 g for 30 min) failed to reveal any abnormal behavior of NZB/W mice as compared to BALB/c or A/J mice. These results do not support the concept that NZB/W mice possess a general immune hyperreactivity or a relative inability to be made tolerant to protein antigens. However, they do not rule out the possibility that these mice have a genetically determined hyperresponsiveness to some antigens, in particular to nuclear antigens

CELLULAR AND HUMORAL RESPONSE TO TRANSPLANTATION ANTIGENS : I. DEVELOPMENT OF ALLOANTIBODY-FORMING CELLS AND CYTOTOXIC LYMPHOCYTES IN THE GRAFT-VERSUS-HOST REACTION

After transfer into heavily-irradiated allogeneic mice, spleen cells were found to produce two types of effector cells directed against the recipient alloantigens, namely alloantibody plaque-forming cells (PFC) and cytotoxic lymphocytes (CL). Both types of effector cells were detectable in vitro by virtue of their lytic effect on target cells carrying the recipient alloantigens. Alloantibody PFC activity was dependent on the presence of an exogenous source of complement and could be inhibited by the addition of heterologous antisera to mouse µ-chain or Fab fragment in the assay system. CL activity was independent of added complement, was not affected by anti-immunoglobulin antisera, but was inhibited by the addition of antibody against target cell alloantigens. Treatment of the transferred spleen cells with anti-θ-serum and complement before in vitro assays for PFC and CL completely abolished the CL activity but had no effect on alloantibody-plaque formation. These results indicate that the two types of effector cells can be differentiated in vitro by virtue of their susceptibility to anti-θ-serum and the mechanisms by which they cause cell lysis

Cytolytic T lymphocyte function is independent of growth phase and position in the mitotic cycle

We have investigated mitotic cell cycle and growth phase regulation of homogeneous cytolytic T lymphocytes (CTL). Two independently derived CTL clones were stained with the DNA-binding dye Hoechst 33342, sorted in a fluorescence-activated cell sorter according to their position in the cell cycle, and then assayed for specific lytic activity using a short-term (30 min) (51)Cr release assay. Results show that lytic activity remained unchanged throughout the cell cycle. Furthermore, there was no significant difference in the lytic activity of CTL clones growing exponentially or arrested in a plateau phase. These results demonstrate that T cell-mediated cytolysis is independent of growth phase and position in the cell cycle

Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer.

We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation. Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05). This reduction correlated with the progression of the disease. The total leucocyte and monocyte population were almost identical in both groups. In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001). Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden. Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion