Considering external information to improve the phylogenetic comparison of microbial communities: a new approach based on constrained Double Principal Coordinates Analysis (cDPCoA).

The use of next-generation sequencing technologies is revolutionizing microbial ecology by allowing a deep phylogenetic coverage of tens to thousands of samples simultaneously. Double Principal Coordinates Analysis (DPCoA) is a multivariate method, developed in community ecology, able to integrate a distance matrix describing differences among species (e.g. phylogenetic distances) in the analysis of a species abundance matrix. This ordination technique has been used recently to describe microbial communities taking into account phylogenetic relatedness. In this work, we extend DPCoA to integrate the information of external variables measured on communities. The constrained Double Principal Coordinates Analysis (cDPCoA) is able to enforce a priori classifications to retrieve subtle differences and (or) remove the effect of confounding factors. We describe the main principles of this new approach and demonstrate its usefulness by providing application examples based on published 16S rRNA gene data sets.

Unexpected male choosiness for mates in a spider.

Sexual selection theory traditionally considers choosiness for mates to be negatively related to intra-sexual competition. Males were classically considered to be the competing, but not the choosy, sex. However, evidence of male choosiness is now accumulating. Male choosiness is expected to increase with an individual's competitive ability, and to decrease as intra-sexual competition increases. However, such predictions have never been tested in field conditions. Here, we explore male mate choice in a spider by studying size-assortative pairing in two natural sites that strongly differ in the level of male-male competition. Unexpectedly, our results demonstrate that mate choice shifts from opportunism to high selectivity as competition between males increases. Males experiencing weak competition did not exhibit size-related mating preferences. By contrast, when competition was intense we found strong size-assortative pairing due to male choice: while larger, more competitive males preferentially paired with larger, more fecund females, smaller males chose smaller females. Thus, we show that mating preferences of males vary with their competitive ability. The distinct preferences exhibited by males of different sizes seem to be an adaptive response to the lower reproductive opportunities arising from increased competition between males.

Two tumor models of curative adoptive chemoimmunotherapy using tumor-infiltrated spleen cells with potent antitumor cytotoxicity stimulated by antigen-sharing tumors.

Previously we have established curative protocols for adoptive chemoimmunotherapy (ACIT) of mice bearing different plasmacytomas that are known to bear cross-reacting antigens: (a) the cure of mice bearing an early-stage, nonpalpable MOPC-315 tumor by a very low dose of cyclophosphamide (10 mg/kg) and cultured MOPC-315-tumor-infiltrated (TI) spleen cells (25 x 10(6)) and (b) the cure of mice bearing a late-stage, relatively drug-resistant, highly metastatic RPC-5 tumor with cyclophosphamide (100 mg/kg) and cultured RPC-5 TI spleen cells (25 x 10(6) - 50 x 10(6)). In both models, the spleen cells were obtained from mice bearing a late-stage tumor and were cultured for 5 days in the presence of polyethyleneglycol 6000 and autochthonous tumor cells as a source of tumor antigen. Here we show that RPC-5 tumor cells could substitute for MOPC-315 tumor cells in the 5-day culture of MOPC-315 TI spleen cells so that they became curative in ACIT for mice bearing an early-stage MOPC-315 tumor. Similarly, MOPC-315 tumor cells could substitute for RPC-5 tumor cells in the 5-day culture of RPC-5 TI spleen cells so that they became curative in ACIT of mice bearing a late-stage RPC-5 tumor. In addition, RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were effective in curing all mice bearing an early-stage MOPC-315 tumor by ACIT. However, MOPC-315 TI spleen cells whether cultured with MOPC-315 or RPC-5 tumor cells, were much less effective than cultured RPC-5 TI spleen cells in curing mice bearing a late-stage RPC-5 tumor by ACIT (although the survival of these mice was extended significantly). Interestingly, whereas RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were as effective as MOPC-315 TI spleen cells cultured under the same conditions in lysing MOPC-315 tumor cells in vitro, MOPC-315 TI spleen cells that had been cultured with either MOPC-315 or RPC-5 tumor cells exerted a much weaker in vitro cytotoxic T lymphocyte activity against RPC-5 tumor cells than did RPC-5 TI spleen cells that had been cultured under the same conditions.

Cure of mice bearing a late-stage, highly metastatic, drug-resistant tumor by adoptive chemoimmunotherapy.

We show here that in contrast to BALB/c mice bearing a late-stage, large MOPC-315 plasmacytoma, BALB/c mice bearing a late-stage, large RPC-5 plasmacytoma were not cured by cyclophosphamide therapy (15, 50, 100 or 200 mg/kg). However, most BALB/c mice bearing a late-stage RPC-5 tumor were cured by cyclophosphamide therapy (100 mg/kg) in conjunction with adoptive immunotherapy using tumor-infiltrated spleen cells (TISpC) that had been cultured with inactivated RPC-5 tumor cells plus polyethylene glycol 6000, even though this protocol was not effective for the therapy of mice bearing a barely palpable, early-stage RPC-5 tumor. Only a few of the mice that were cured of a late-stage RPC-5 tumor following adoptive chemoimmunotherapy (ACIT) were resistant to a subsequent challenge with RPC-5 tumor cells. However, the challenged mice that had developed progressively growing tumors could then be cured by cyclophosphamide alone when the tumor became large, even though this treatment was not curative for mice bearing a tumor of similar size but not previously treated by ACIT. Thus, the cure by ACIT of BALB/c mice bearing a lethal, late-stage RPC-5 tumor with extensive metastases provides a novel experimental tumor model for investigating the mechanisms by which a chemotherapeutic drug and adoptive cellular immunotherapy can cooperate in causing the complete regression of a large tumor load.

Advantages of adoptive chemoimmunotherapy with polyethylene glycol-cultured, antigen-activated, tumor-infiltrated spleen cells for the complete eradication of lethal MOPC-315 plasmacytomas.

The incorporation of polyethylene glycol-6000 (PEG) into the culture media of tumor-infiltrated spleen cells (TISpC) and MOPC-315 stimulator tumor cells at a responder to stimulator cell ratio of 30/1 had been shown to lead to the appearance of CD8+ T-cells that were effective in adoptive chemoimmunotherapy (ACIT) of mice bearing a barely palpable MOPC-315 tumor (J. A. Wise, M. B. Mokyr, and S. Dray, Cancer Res., 49:3613-3619, 1989). Here we show that in the presence of substantially fewer added stimulator tumor cells (responder to stimulator cell ratio, 100/1), the inclusion of PEG in the cultures of TISpC also enhanced the appearance of cells that were highly effective in curing such mice by ACIT. Moreover, these PEG-cultured TISpC were more effective in ACIT than TISpC cultured in the presence of an optimal concentration of recombinant interleukin-2 (60 IU/ml). The potency of the tumor-eradicating activity of the PEG-cultured TISpC in ACIT was further illustrated by their ability to cause the complete regression of a large (20-22 mm) s.c. MOPC-315 tumor in conjunction with a dose of drug that by itself did not cause tumor regression. PEG-cultured TISpC that were effective against MOPC-315 tumor cells in an antigen-specific manner. In fact, PEG-cultured TISpC were more effective than recombinant interleukin-2-cultured TISpC, not only in ACIT, but also in their ability to lyse MOPC-315 tumor cells in vitro. Thus, a direct specific lytic activity against the tumor by cytotoxic T-lymphocytes is the apparent mechanism through which the complete regression of the large tumor burden is brought about by the PEG-cultured TISpC. Finally, we suggest that the incorporation of PEG to render ineffective lymphoid cells effective in ACIT may offer some advantages compared with the incorporation of recombinant interleukin-2 and may be suitable for protocols to generate human cytotoxic cells for cancer therapy when there are relatively low numbers of available tumor cells.

Serine proteinase inhibitors produced by human melanoma cell lines.

The human melanoma cell lines M21 and MSM-M2 are shown to produce two similar competitive inhibitors of trypsin, a serine proteinase. These proteinase inhibitors inhibited the serine proteinases trypsin and kallikrein with similar efficiency but did not inhibit plasmin (a serine proteinase) or papain (a thiol proteinase). Active synthesis of the inhibitors during cell culture was indicated by the requirement for cell viability, the increase in inhibitory activity of the supernatant with time, and the incorporation of 35S-methionine into the inhibitors. The two inhibitors were stable to heat (70 degrees C) and extremes of pH. Their molecular weights were estimated at 670 and 250 kD, respectively. A screening of the supernatants of five other human melanoma cell lines by HPLC showed that they all released a similar trypsin inhibitory factor not detected in human or bovine serum. The isolation of these proteinase inhibitors facilitates a study of their putative role in tumor growth.

Inhibitors of lymphocyte activation secreted by human melanoma cell lines.

The concentrated supernatants of nine human melanoma cell line cultures were analyzed for the presence of factors that inhibit in vitro immunological reactions. All cell lines secreted a factor that inhibited LPS-induced proliferation of murine B cells; eight cell lines released a factor that inhibited PHA-induced proliferation of murine T cells; all of the three cell lines investigated secreted a factor that inhibited the allogeneic stimulation of BALB/c spleen cells by mitomycin-C-treated C57BL spleen cells. Further analysis of the M21 cell supernatant indicated that its continuous presence was required for the inhibition of the PHA but not of the LPS response suggesting a different mechanism of action. High levels of PHA, but not of LPS, could overcome the inhibitory effect of M21 supernatants. Fractionation of M21 supernatants by sucrose gradient centrifugation, DEAE chromatography and gel filtration suggested that the anti-PHA and the anti-LPS activities were due to different factors. These factors differed from a serine proteinase inhibitor that is also released by M21 cells. Since these inhibitory factors may have a role in protecting a melanoma tumor from attack by the immune system of the host, their consideration may be helpful in designing protocols for therapy which include methods to boost the antitumor responses of the host.

Lysis of antigenically unrelated tumor cells mediated by Lyt 2+ splenic T-cells from melphalan-cured MOPC-315 tumor bearers.

We have previously shown that mice cured of a large MOPC-315 tumor following low-dose melphalan (L-phenylalanine mustard, L-PAM) therapy can exert, upon challenge with MOPC-315 tumor cells, an antitumor effect against innocent bystander tumor cells present within the same tumor site (Barker, E., and Mokyr, M.B. Cancer Immunol. Immunother., 25: 215-224, 1987). Here we show that T-cells are important for the MOPC-315-induced rejection of MOPC-104E tumor cells present within the same site. To further characterize the innocent bystander killing activity exerted by L-PAM-cured MOPC-315 tumor bearers upon stimulation with MOPC-315 tumor cells, we established the in vitro conditions under which lymphoid cells from L-PAM-cured MOPC-315 tumor bearers can exert an antitumor effect against innocent bystanders. Specifically, we established that spleen cells from mice that just completed the rejection of a large MOPC-315 tumor following low-dose L-PAM therapy can, upon stimulation with MOPC-315 tumor cells, bring about the killing of antigenically unrelated tumor cells in a 12-h 51Cr release assay. The magnitude of lysis of EL4 and WEHI 22.1 tumor cells by MOPC-315 in vitro-immunized (IVI) spleen cells from L-PAM-cured MOPC-315 tumor bearers can be substantially enhanced upon reexposure of the spleen cells to MOPC-315-associated antigens during the 12-h 51Cr release assay. The lysis of innocent bystander tumor cells by these MOPC-315-IVI spleen cells was found to be mediated by T-cells of the Lyt 2 and not the L3T4 phenotype. These Lyt 2+ T-cells did not appear to mediate their lytic activity for innocent bystander tumor cells via effector macrophages, since a drastic reduction in macrophage frequency among the MOPC-315-IVI spleen cells just prior to assessing the lytic activity of the spleen cells did not reduce, but actually enhanced, the magnitude of EL4 lysis. In addition, a Lyt 2+ T-cell clone derived from mice cured of a large MOPC-315 tumor by a low dose of drug was capable, upon stimulation with MOPC-315 tumor cells, of exerting a potent lytic effect against EL4 and WEHI 22.1 tumor cells in the 12-h 51Cr release assay. Thus, Lyt 2+ T-cells independent of effector macrophages are responsible for lysis of innocent bystander tumor cells by MOPC-315-IVI spleen cells from L-PAM-cured MOPC-315 tumor bearers.

Enhancement of the effectiveness of Lyt-2+ T-cells for adoptive chemoimmunotherapy by short-term exposure of tumor-bearer spleen cells to polyethylene glycol and/or melphalan.

Uncultured tumor-infiltrated spleen cells (TISpC) from mice bearing large (20-22 mm) s.c. MOPC-315 plasmacytomas were previously shown to be ineffective in bringing about the cure of mice bearing a nonpalpable (Day 4) tumor that had been treated with a subcurative dose (10 mg/kg) of cyclophosphamide (i.e., adoptive chemoimmunotherapy, ACIT) (M. B. Mokyr, J. C. D. Hengst, and S. Dray, Cancer Res., 42:974-979, 1982). Here we show that TISpC cultured for 5 days in the presence of inactivated MOPC-315 stimulator cells acquire some effectiveness in curing mice by ACIT, and this effectiveness is greatly enhanced if polyethylene glycol 6000 (PEG) is also added to the culture. The Lyt 2+ T-cells, and not the L3T4+ T-cells, are responsible for the effectiveness of the cultured TISpC in ACIT. In fact, the L3T4+ T-cells are apparently not required even during culture of TISpC for the generation of Lyt 2+ T-cells effective in ACIT. Although the TISpC cultured with MOPC-315 cells and PEG contained approximately twice as many Lyt 2+ cells as did TISpC cultured without PEG, the increase in the activity of the former cells is not due simply to the increase in the percentage of Lyt 2+ cells, but is most likely due to an increase in the percentage and/or activity of Lyt 2+ cells with specificity for MOPC-315-associated antigens. The effectiveness of TISpC cultured with MOPC-315 stimulator cells and PEG in ACIT can be enhanced even further by pretreatment of these cells with the immunomodulating agent melphalan (0.5 nmol/ml) prior to culture initiation. Thus, the above methods of culture render ineffective lymphoid cells effective in ACIT and are suitable for evaluation in protocols for human cancer therapy.

Cyclophosphamide and melphalan as immunopotentiating agents in cancer therapy.

The murine plasmacytoma, MOPC-315, has been used as a tumor model to investigate the immunopotentiating effect of a low dose of cyclophosphamide (CY) or melphalan (L-PAM). Each drug was shown to shift the balance in mice bearing a late-stage tumor from a state of immunosuppression to that of potent T-cell-dependent antitumor immunity against tumor-associated antigens. The resultant immunity eradicated the extensive tumor burden not already eradicated by the direct tumoricidal activity of the drug and brought about the cure of the mice. The immunity responsible for tumor eradication, as well as the immunity responsible for the resistance of the cured mice to further tumor challenge, was mediated by the Lyt 2 subset of T-cells which contains cytotoxic T-cells. The principle of using a low dose of drug to selectively decrease suppressor cell activity so as to allow the development of antitumor immunity with the aid of autologous tumor vaccine or interleukin-2 has been exploited successfully by clinicians in therapeutic protocols for human melanoma.

Effect of low-dose cyclophosphamide therapy on specific and nonspecific T cell-dependent immune responses of spleen cells from mice bearing large MOPC-315 plasmacytomas.

Some T cell-dependent immune parameters were examined in mice bearing a large MOPC-315 plasmacytoma before and after treatment with a low dose (15 mg/kg) of CY. Prior to CY therapy, spleen cells from mice bearing a large MOPC-315 tumor were depressed in their ability to generate an in vitro cytotoxic response to the MOPC-315 tumor, to a different syngeneic plasmacytoma, MOPC-104E, and to an allogeneic thymoma, EL4. The spleen cells of these mice were also depressed in their ability to proliferate in response to the T cell mitogen PHA. Following CY therapy, the spleen cells generated an enhanced anti-MOPC-315 cytotoxic response by day 2, and the level of this response continued to increase so that by day 7, it was greatly enhanced and was much greater than the response of normal spleen cells. The recovery of the cytotoxic responsiveness to the antigenically related MOPC-104E tumor after CY therapy followed a similar pattern. In contrast, the spleen cells of these animals remained depressed in their cytotoxic response to the antigenically unrelated EL4 thymoma for at least 11 days after CY therapy. Although the anti-EL4 response recovered by day 14, the level of antitumor cytotoxicity generated did not exceed that generated by normal spleen cells. The PHA response remained greatly depressed in CY-treated MOPC-315 tumor bearers, even 14 days after the chemotherapy. Thus, at a time following low-dose CY therapy, when potent T cell-dependent antiplasmacytoma immunity had completed the eradication of a large MOPC-315 tumor burden not eliminated through the direct effect of the drug, the T cell-dependent response to an unrelated tumor and to PHA remained depressed.

Tumoricidal and immunomodulatory activities of drugs and implications for therapy of mice bearing a late stage MOPC-315 plasmacytoma.

The effectiveness of a relatively low dose of cyclophosphamide (15 mg/kg CY), melphalan (2.5 mg/kg L-PAM) or the monofunctional form of CY (150 mg/kg MoCY) for the cure of mice bearing a large primary s.c. MOPC-315 tumor and extensive metastases has been shown to be dependent on the cooperation of the drugs' tumoricidal activity with T-cell-dependent antitumor immunity, the latter facilitated by the drug's immunomodulatory activity. Here, we have compared the curative effectiveness of three additional drugs: methyl nitrosourea (MNU), hydroxyurea (OH-urea) and bis-chloroethyl nitrosourea (BCNU). Among these drugs, only a relatively low dose of BCNU (15-20 mg/kg) was effective in curing most mice (85%) bearing a large, late stage tumor. A higher dose of BCNU (40 mg/kg, LD10) was much less effective. After an optimal dose of BCNU, the proliferative capacity of the tumor cells 24 h after therapy was reduced by greater than 97%. However, viable tumorigenic cells were still present in the primary tumor and enhanced T-cell-dependent antitumor immunity was necessary for their eradication. The cured mice were resistant to tumor rechallenge. When a low curative dose of L-PAM was followed by OH-urea, the therapeutic effectiveness was not affected, but when this dose of L-PAM was followed by a high nontoxic dose of MNU (100-150 mg/kg), the therapeutic effectiveness was diminished even though MNU was highly tumoricidal (i.e. greater than 99% inhibition of proliferative activity). Thus, BCNU appears to be similar to CY, L-PAM and MoCY in its mechanism of MOPC-315 tumor eradication. The alkylating activity of CY, L-PAM, MoCY and BCNU appears to be critical for their combined tumoricidal and immunomodulatory effects. Since BCNU is the simplest of these four drugs with respect to metabolic pathway, a further study with BCNU and related constructs may shed some light on the biochemical mechanisms of their mode of action. At least one reason for the ineffectiveness of OH-urea or MNU at either low or nontoxic high doses was poor tumoricidal or immunomodulatory activity, respectively. Thus, it seems important to consider both the tumoricidal and immunomodulatory activities of drugs when developing regimens for effective chemotherapy.

A serine proteinase inhibitor produced by an HTLV I virus-transformed human T lymphocyte line.

A previous report from our laboratory indicated that a proteinase inhibitor is produced by rabbit T lymphocytes. We now report that a human T cell line, C91/PL, produces a proteinase inhibitor which inhibits the enzymatic activity of trypsin and kallikrein. This newly identified proteinase inhibitor (LPI 1) did not inhibit the enzymatic activity of four other serine proteinases (thrombin, plasmin, chymotrypsin, or pancreatic elastase), a thiol proteinase (papain), or a carboxyl proteinase (pepsin). Active synthesis of LPI 1 by the C91/PL cell line was shown by the appearance of similar levels of inhibitory activity in sequential cell supernatants, lack of appearance of inhibitor in supernatants of cells killed by heat or sodium azide or of viable cells in the presence of cyclohexamide, and incorporation of a radiolabeled amino acid into newly synthesized inhibitor. Although both the inhibitor of rabbit origin and of human origin are proteins produced by T cells and have similar inhibitory specificity, important differences were observed: LPI 1 is sensitive to boiling and the two inhibitors migrate differently upon electrophoresis in substrate-containing polyacrylamide gel. Furthermore, LPI 1 was produced by a cell line of the T4 phenotype which had been established by in vitro viral transformation of human cord blood lymphocytes with HTLV 1 whereas the inhibitor of rabbit origin was produced by normal splenic T cells. Three other human T cell lines of the T4 phenotype, MOLT-13, KE-37, and HPB-ALL, from patients with acute lymphoblastic leukemia did not produce a proteinase inhibitor. Thus, the production of proteinase inhibitors does not appear to be a general characteristic of human T cell lines nor of the T4 subset. Proteinase inhibitors produced by T cells may have an immunoregulatory role in proteinase-mediated physiological processes.

Interplay between the toxic effects of anticancer drugs and host antitumor immunity in cancer therapy.

The results accumulated thus far illustrate that the therapeutic efficacy of many anticancer drugs depends not only on the direct tumoricidal/tumoristatic activity of the drug but also on the contribution to tumor eradication of antitumor immunity which emerges after the chemotherapy. When a low dose of anticancer drug is employed, it reduces the tumor burden to a lesser extent than a high dose of drug. Consequently, in order for the low dose of drug to be as effective as a high dose of drug, antitumor immunity has to control a larger tumor burden than that controlled by the immune system following high-dose chemotherapy. This was shown to happen in several experimental tumor models wherein the low dose of drug greatly potentiated host antitumor immunity while the high dose of drug either potentiated host antitumor immunity to a lesser extent, did not potentiate it at all, or actually exerted a suppressive effect on host antitumor immunity. As a result of the immunopotentiating activity of the low dose of drug, tumor-bearing animals which did not exhibit concomitant antitumor immunity due to the inhibitory activity of suppressor cells, developed a very potent antitumor immunity shortly after the chemotherapy. The immunopotentiating effect of the low dose of drug was attributed in these situations to drug-mediated selective elimination of suppressor cell activity and possibly also to drug-mediated enhancement in the activity of T cells of the helper phenotype through elevation in IL-2 production. Antitumor immunity can also facilitate the therapeutic effectiveness of high-dose chemotherapy. Therefore, it is important to determine the conditions under which high-dose chemotherapy can also potentiate host antitumor immunity. From the rodent data available, the picture emerges that the maturity of antitumor immunity at the time of the chemotherapy is an important factor in determining if the high dose of drug leads to immunosuppression or immunopotentiation. When a high dose of drug is administered to tumor bearers with a fully developed antitumor immune response, it is more likely to lead to potentiation of host antitumor immunity than when the high dose of drug is administered to tumor bearers that have not yet achieved the full maturity of their antitumor immune response. In order to achieve the maximal enhancement of antitumor immunity in hitherto immunosuppressed tumor bearers, the suppressor-cell pool should be more sensitive to the toxic effects of the anticancer drugs than are cells involved in immune-tumor eradication.(ABSTRACT TRUNCATED AT 400 WORDS)

Ability of cyclophosphamide in the absence of cross-linking activity to exert the immunomodulatory effect required for the cure of mice bearing a large MOPC-315 tumor.

We have previously shown that mice bearing a late-stage, large primary MOPC-315 plasmacytoma and extensive metastases can be cured by a low dose of the bifunctional alkylating drug, cyclophosphamide (BiCY) (J.C.D. Hengst et al., Cancer Res., 40: 2135-2141, 1980). Here we show that therapy with the monofunctional form of cyclophosphamide (MoCY) can also cure such mice. However, a dose of at least 150 mg of MoCY per kg is required to approximate the curative effectiveness of the lowest curative dose of BiCY, i.e., 15 mg/kg. This need for a 10-fold higher dose of MoCY is due, at least in part, to the 10-fold lower direct tumoricidal and/or tumoristatic activity of MoCY compared to BiCY. Consequently, a 10-fold higher dose of MoCY is required to directly reduce the tumor burden to the level reduced by 15 mg of BiCY per kg. Other than dose, the therapy of the mice with 150 mg of MoCY per kg was similar in its essential features to that shown previously for therapy with 15 mg of BiCY per kg (J.C.D. Hengst et al., Cancer Res., 40: 2135-2141, 1980; J.C.D. Hengst et al., Cancer Res., 41:2163-2167, 1981; Q-W. Ye et al., Cancer Immunol. Immunother., 16:162-169, 1984; Q-W. Ye and M.B. Mokyr, Cancer Res., 44: 3873-3879, 1984; M.B. Mokyr and S. Dray, Cancer Res., 43: 3112-3119, 1983), namely: (a) the drug does not directly eradicate all tumor cells; (b) host T-cell-dependent antitumor immunity is also required for the curative effect; (c) the therapy of tumor bearers leads to the rapid appearance of an augmented antitumor immune potential in their hitherto immunosuppressed spleen; and (d) the cured mice are resistant to a subsequent challenge with at least 300-fold the minimal lethal tumor dose. Thus, cross-linking is not an essential property for the immunomodulatory activity of BiCY nor for its direct antitumor effect. However, in the presence of cross-linking activity, a much lower dose of drug is effective.

Partial amino acid sequence and genetic control of latent a2 allotype induced in rabbits by immunization with anti-a2 antibody.

We have shown that after immunization of homozygous a1 rabbits of the B immunoglobulin (Ig) heavy chain haplotype with anti-a2 antibody (Ab) a population of molecules appears that has all of the serologic characteristics of the a2 allotype. We have now isolated these putative latent a2 molecules, have separated the heavy chains, and after enzymatic deblocking, have determined the first 19 N-terminal amino acids. For all eight allotype-associated residues, these putative latent a2 molecules have the amino acid residues typical of a2 allotype. As expected, the preimmune IgG from this a1a1 rabbit has the amino acids typical of the a1 allotype. Thus by partial amino acid sequence analysis, we provide additional evidence that the latent a2 allotype can be induced in a1a1 rabbits of the B heavy chain haplotype by immunization with anti-a2 Ab. Rabbits of other heavy chain haplotypes were also immunized with anti-a2 Ab and were tested for their ability to synthesize latent a2 allotype. Thus far, a1a1 rabbits of the A, B, C, and I heavy chain haplotypes all synthesize latent a2 allotype. In contrast, a3a3 rabbits of the G and H heavy chain haplotypes did not synthesize latent a2 allotype.

A low molecular weight proteinase inhibitor produced by T lymphocytes.

A low molecular weight (MW) proteinase inhibitor, between 6500 and 21,500 MW, appeared in the supernatant of rabbit spleen cells cultured at high density for 24 hr. The inhibitor inhibited the enzymatic activity of trypsin for both a high MW natural substrate, fibrinogen, and for a low MW artificial substrate, Chromozym TRY. The low MW proteinase inhibitor is protein in nature and is different, in terms of specificity for enzymes, MW and sensitivity to different physical or chemical treatments, from aprotinin, a low MW proteinase inhibitor (6500 MW) of bovine origin, and from the soybean trypsin inhibitor, a relatively high MW proteinase inhibitor (21,500 MW). The inhibitor was found in the supernatant of purified T cells but not B cells, and its production was increased in the presence of an optimal concentration of Con A. The possibility that this proteinase inhibitor has a role in the regulation of trypsin-like proteinases involved to the immune response remains to be investigated.

Allogeneic lymphocyte stimulation in rabbits: induction of a low MW inhibitor for trypsin and for a concurrently induced alpha-macroglobulin-proteinase complex.

We have shown previously that the i.v. inoculation of allogeneic lymph node cells in rabbits induces the appearance in the serum of an alpha M-serine proteinase complex which behaves in an Ig-turnover assay as any polyclonal B-cell activator (PBA), and that this PBA activity is due to the enzyme. Here, we show that the allogeneic stimulation also induces the appearance in the low molecular weight fraction of the serum (1000-110,000 MW) of an inhibitor which blocks the PBA activity of the complex without affecting the PBA activity of LPS or dextran sulphate. The inhibitor blocked the ability of the enzyme associated with alpha M to degrade Chromozym TRY, a low MW trypsin substrate. The inhibitor also blocked the enzymatic activity of trypsin for large as well as for low MW substrates. Thus, allogeneic stimulation in vivo results in the production, not only of an alpha M-proteinase complex, but also of an inhibitor for this proteinase as well as for trypsin. The appearance of the inhibitor, along with the alpha M-serine proteinase complex as a result of allogeneic stimulation in rabbits, is of interest since a similar alpha M-serine proteinase complex and inhibitor may appear in the serum of patients with rheumatoid arthritis.

Allotypic and isotypic specificities of rabbit IgM: localization to the Fab mu or Fc mu fragments.

IgM from trypanosome-infected rabbits was digested with trypsin under different conditions to obtain Fab mu or Fc5 mu fragments suitable for analysis with anti-allotype and anti-isotype antibodies. The Fab mu but not the Fc5 mu fragment was shown to have the n-locus allotypic specificities, n80, n81, n82, n83 and n87, characteristic of the IgM class of immunoglobulins. Thus, the n82 and n83 allotypic specificities, conformationally dependent on the a VH locus for expression, and the n80, n81 and n87 allotypic specificities, independent of the a VH locus for expression, are in either the CH1 or CH2 domain of IgM heavy chains. In addition, two high-affinity mouse monoclonal antibodies (MoAbs) specific for IgM and able to bind IgM in direct-binding radioimmunoassays were produced and characterized. One MoAb (3C1) was specific for an isotypic determinant (epitope) in the Fab mu fragment, presumably in the CH1 or CH2 domain, whereas another MoAb (8C2) was specific for an isotypic epitope in the Fc5 mu fragment, presumably in the CH3 or CH4 domain. The proximity of the n-locus allotypic specificities (CH1 or CH2 domain) to the VH domain is consistent with the finding that some IgM allotypic specificities are expressed only in conjunction with certain a VH locus allotypic specificities.

Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor.

Exposure of MOPC-315 cells from the primary tumor nodule to a low concentration (0.5 nmol/ml) of melphalan (L-phenylalanine mustard; L-PAM) rendered the tumor cells capable of bringing about the generation of a potent primary antitumor cytotoxic response. Accordingly, the level of antitumor cytotoxicity generated by normal spleen cells immunized in vitro with L-PAm-treated tumor cells was at least five-fold greater than the level generated in response to untreated tumor cells. The marked superiority of L-PAM-treated tumor cells over untreated tumor cells in bringing about the generation of antitumor cytotoxicity was evident over a wide range of responder to stimulator cell ratios. The higher level of antitumor cytotoxicity exhibited by normal spleen cells immunized with L-PAM-treated tumor cells as compared with untreated tumor cells was not merely the result of direct drug-mediated tumoricidal activity, thereby reducing the number of tumor cells present which can act as cold target cell inhibitors during the 51Cr release assay. This is apparent from the observation that the level of antitumor cytotoxicity generated in response to a given percentage of stimulator tumor cells pretreated with 0.5 nmol L-PAM/ml, a drug concentration associated with retention of 60% tumor cell proliferative capacity, is substantially greater than that generated in response to less than half that percentage of untreated stimulator tumor cells. Moreover, stimulator tumor cells exposed to a fully antiproliferative concentration of L-PAM brought about the generation of a higher level of antitumor cytotoxicity than stimulator tumor cells exposed to mitomycin C at a concentration which inhibited the proliferation of the tumor cells to the same extent as the L-PAM. A low concentration of L-PAM which was effective in rendering isolated tumor cells from the primary tumor nodule capable of bringing about the generation of antitumor cytotoxicity was also effective in inducing the appearance of potent antitumor immune potential in tumor bearer splenic cells containing metastatic tumor cells.