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Peer review is believed to be important in maintaining the quality and integrity of research in academic endeavors. Recently, the value of the current peer review process, which is more than 100 years old has come into question. In the field of hematopoietic cell transplantation (HCT), peer review was unable to prevent the publication of the largest and most notorious scientific fraud in our field. In order to assess how the HCT community views and how engaged it is with the peer review process, the American Society of Blood and Marrow Transplantation conducted a survey of all of its members in 2014. The survey was sent to all active members through multiple email communications in August and September 2014. Of a total of 1183 members, 149 responded. Almost all of the respondents had participated in the peer review process, with few respondents declining ever to review manuscripts. The most common cause for declining review requests was lack of time. Most respondents (68%) thought that the current peer review process was relatively fair and unbiased, whereas only 9% of the respondents stated that they did not believe in the peer review process. In conclusion, among the respondents of this survey most felt the peer review process to be valuable and fair, however, the lack of response suggests that further study into improving the peer review process in the field of HCT is warranted in the era of electronic publishing and communication.

Graft-vs-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. Experimentally, lethal GVHD can be induced in MHC-matched strain combinations differing in expression of multiple minor histocompatibility Ags (miHA). Recently, the GVHD potential of C57BL/6By (B6) T cells in irradiated BALB.B (both H2b) and related CXB recombinant inbred strains of mice has been studied to determine the scope of the response to miHA in vivo and how it compared with CTL responses to immunodominant miHA in vitro. The GVHD response in these strain combinations appeared to be limited to a few Ags, yet there was no correlation of these miHA with that of in vitro CTL responses. To further investigate the role of CD8+ T cells in GVHD, we analyzed positively selected miHA-specific donor CD8+ thoracic duct lymphocytes (TDL) collected from irradiated BALB.B and CXBE mice, 5 to 6 days after transplantation of B6 T cells. Flow cytometric analysis of B6→BALB.B TDL did not indicate expansion of any particular TCR Vβ family, whereas Vβ10 and Vβ14 families were significantly expanded in the B6->CXBE TDL. However, PCR-based complementarity-determining region 3 size spectratyping revealed overlapping involvement of donor Vβ1, 6, 8, 9, 10, and 14 families in both BALB.B and CXBE recipients and unique utilization of the Vβ4 family in BALB.B mice, suggesting oligoclonal T cell responses to a limited number of miHA. In addition, the injection of CD8+Vβ14+ B6 T cells into irradiated BALB.B and CXBE mice induced lethal GVHD, confirming the involvement of miHA-specific T cells within an individual Vβ family.

This study was undertaken to distinguish between several mechanisms responsible for graft-vs-host disease (GVHD) protection in anti-CD3epsilonF(ab')2 fragment (Fr)-treated recipients: TCR down-modulation, deletion, failure of expansion, or anergy induction. To quantify alloreactive T cell expansion and function, thoracic duct lymphocytes (TDL) were analyzed. Sixfold fewer donor TDL T cells were recoverable from anti-CD3epsilonF(ab')2 Fr as compared with irrelevant F(ab')2 Fr-treated recipients at the time of peak T cell expansion in vivo. Kinetic analysis revealed that donor T cell expansion was inhibited and not simply delayed by anti-CD3epsilonF(ab')2 Fr. Similar proportions of TDL T cells in irrelevant and anti-CD3epsilonF(ab')2 Fr were undergoing apoptosis. Although TCR modulation was observed, donor TDL T cells had intact anti-host alloresponses as compared with irrelevant F(ab')2 Fr-treated recipients. Because donor CD4+ T cells are primarily responsible for GVHD in this model, an adoptive transfer system was used in which the function and kinetics of expansion of OVA-specific CD4+ TCR transgenic cells could be physically tracked. Relevant Fr severely blunted CD4+ TCR transgenic T cell clonal expansion after OVA administration. Nonviable transgenic and nontransgenic T cells were proportionally similar in OVA-pulsed recipients, regardless of whether relevant or irrelevant F(ab')2 Fr were given. After discontinuing Fr, transgenic T cells were found to have intact in vitro OVA-specific responses. Our current and previous results suggest that reduced donor T cell expansion and T cell depletion both contribute to GVHD protection by anti-CD3epsilonF(ab')2 Fr. These data have implications for designing therapeutic approaches directed toward TCR targeting in humans.

Prophylactic therapy with 5′-dCACGTTGAGGGGCAT phosphorothioate, (2.5 nmol/hr) strongly inhibited tumorigenesis in Eμ-myc transgenic mice, and ablated spleen cell MYC antigen. However, the anti-c-myc DNA also stimulated proliferation of spleen cells in vitro, though much less so than a positive CG motif control, 5′-dGCATGACGTTGAGCT.

In vitro CTL responses to multiple minor histocompatibility Ags (miHA) are governed by immunodominance as demonstrated in the C57BL/6By (B6) anti-BALB.B strain combination. Immunodominance was also demonstrated to be operative in graft-vs-host disease (GVHD) responses against BALB.B-derived miHA following transplantation of B6 T cells into irradiated recipients of both the BALB.B and CXB recombinant inbred strains. The hierarchy of in vivo and in vitro T cell responses to miHA differed. GVHD did not develop in CXBG and CXBK mice, which express immunodominant miHA for CTL generation, whereas disease occurred in the BALB.B, CXBE, CXBI, and CXBJ mouse strains. Previous results demonstrated that B6 CD4+ T cells provide helper function for CD8+ T cells involved in GVHD responses in the BALB.B, CXBE, and CXBI strains. CD4+ T cells alone were mediators of GVHD in all strains except CXBE. This study analyzed the TCR Vbeta repertoires of CD4+ thoracic duct lymphocytes (TDL) collected during the initial stages of GVHD in the B6-->BALB.B and B6-->CXBE strain combinations. Positively selected CD4+ TDL from the B6-->BALB.B (B6(+BALB.B)) combination exhibited marked expansion in the TCR Vbeta6+ and Vbeta8.1/8.2+ families, as well as smaller increases in the Vbeta7 and Vbeta9 families. CD4+ TDL from the B6-->CXBE (B6(+CXBE)) combination displayed expansions in only the Vbeta7+ and Vbeta9+ families. These data suggest that B6 CD4+ T cells can recognize a limited number of immunodominant miHA during GVHD induction and that in both BALB.B and CXBE recipients, the TCR Vbeta repertoires partially overlap.

Alloreactive T cells require costimulatory signals via CD40 ligand (CD40L). The tissue-destructive properties of allogeneic CD4+ but not CD8+ T cells were inhibited by anti-CD40L mAb. Fewer CD4+ thoracic duct lymphocytes (TDL) were obtained in mAb-treated recipients. Kinetic studies revealed that CD4+ T cell expansion was reduced or delayed which may account, in part, for the partial graft-vs-host disease protective effect of anti-CD40L mAb. TDL were found to have diminished anti-host-specific proliferative responses. The frequency of donor TDL and splenocytes that expressed the Th1 cytokines IL-2, IL-12 p40, and IFN-gamma mRNA was markedly diminished in mAb-treated recipients, demonstrating that Th1-driven alloresponses were susceptible to CD40L targeting. Perforin mRNA-expressing T cells were undetectable in mAb-treated recipients, consistent with reduced in vivo lethality after the adoptive transfer of allogeneic CD4+ T cells. Similar findings were observed in both B cell-replete or -deficient recipients, indicating that allogeneic T cell expansion and priming can be sustained by a non-B cell, CD40+ host cell population. Mice receiving CD40L-deficient allogeneic CD4+ T cells had survival rates comparable to the rates of those given anti-CD40L mAb treatment. Because anti-CD40L mAb also was found to prevent host anti-donor-mediated marrow allograft rejection, in vivo blockade of CD40L-CD40 interactions may provide a highly beneficial approach to improving the outcome of allogeneic bone marrow transplantation.

A structure-based design approach was used to develop a cyclized peptide analog of the murine CD4-CDR3-like region as a potential inhibitor of autoimmune CD4+ T cells responsible for the pathogenesis of experimental allergic encephalomyelitis (EAE). Our results indicate that this peptide, referred to as rD-mPGPtide, is able to significantly inhibit the clinical and pathologic symptoms of EAE in the SJL mouse model when administered on day 12 of induction. The optimum effective dosage range for the peptide, injected i.v., was between 0.125 and 0.5 mg and dosages of as high as 5 mg had no observable toxic effects. Treated mice had normal levels of lymphocytes less than 2 wk later and exhibited normal in vitro primary responses to alloantigen and secondary responses to keyhole limpet hemocyanin Ag. The specificity of the rD-mPGPtide treatment for autoreactive T cells was demonstrated by inhibiting proteolipid protein (p139-151)-induced EAE and finding that the lymph node T cells from these mice had suppressed responses to this Ag, but normal responses to alloantigen or other nominal Ag. Importantly, rD-mPGPtide was found to be effective on secondary T cell responses in an EAE rechallenge situation and was able to establish conditions for long-term resistance to further Ag exposure. Analysis of the cytokine profile of responding T cells during late effector stages of disease revealed that the levels of IFN-gamma and IL-4 are significantly reduced in rD-mPGPtide-treated mice. These results strongly suggest that the administration of a CD4-CDR3 peptide analog is an effective therapeutic approach for the inhibition of the CD4+ T cell-mediated autoimmune response in EAE.

Pertussis toxin (Ptx), an important adjuvant for inducing certain organ-specific autoimmune diseases in mice, exerts multiple effects upon the immune system. In addition to its adjuvant effects, which include enhancement of delayed-type hypersensitivity and increased antibody production. Ptx elicits a marked lymphocytosis with a concomitant decrease in thymic weight. In vitro studies indicate that Ptx acts directly on thymocytes and that both susceptible and resistant populations exist. It is believed that these susceptible cells are released into the circulation and account, in part, for the T cell component of the lymphocytosis. We have used flow cytometry to analyze the CD4, CD8, and Thy-1 phenotypes of thymic and peripheral T cells from Ptx-treated mice. In the thymus, there is a dramatic decrease in the number of CD4+CD8+ (double positive) cells at all doses tested (0.25, 0.50, and 1.0 microgram) by day 4 after Ptx treatment. The double negative and single positive populations remain relatively constant. Analysis of Thy-1 expression reveals a significant reduction in Thy-1hi thymocytes, with little change in the Thy-1lo population. Thus Ptx primarily affects and depletes, in a dose-dependent fashion, thymic T cells with an immature phenotype. These results mimic those of corticosteroids, although neither prior adrenalectomy nor treatment with the antiglucocorticoid RU486 are able to prevent the effects of Ptx. In the periphery of Ptx-treated animals, the relative increase in the number of CD4+ T cells is more than that of CD8+ T cells. Double positive and Thy-1hi cells cannot be detected in appreciable numbers. These results are consistent with the concept that Ptx may drive immature thymocytes through accelerated maturation for release into the periphery as single positive, predominantly CD4+, Thy-1lo cells. Increased numbers of such cells may in part account for the immunopotentiating effects of Ptx, particularly as they relate to the induction of organ-specific autoimmune disease. Treatment with purified Ptx beta-oligomer fails to elicit any of the responses described above, indicating that the holotoxin is required for such activities.

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD.

Detailed information was sought on the capacity of purified B6 L3T4+ cells to elicit lethal graft-versus-host disease (GVHD) in irradiated class II-different class I-identical (C57BL/6 (B6) x bm 12)F1 hosts. When B6 L3T4+ cells were transferred in small doses (10(5) to 10(6) together with donor bone marrow (BM) cells, the recipients all developed acute lethal GVHD and most of the mice died within 2 wk, probably from gut damage; this syndrome was conspicuous only in mice treated with very heavy irradiation, i.e., 1000 rad. In marked contrast to L3T4+ cells given in small doses, transfer of large doses of B6 L3T4+ cells to heavily irradiated (B6 x bm 12)F1 hosts paradoxically resulted in only limited mortality: most of the recipients survived for greater than 6 mo and manifested little or no evidence of ill health. It is suggested that the capacity of large doses of L3T4+ cells to protect mice against lethal GVHD is a reflection of T helper function: the cellular immunity provided by the donor L3T4+ cells enables the host to repel pathogens entering through damaged mucosal surfaces, with the result that GVHD becomes sublethal. The protective function of L3T4+ cells in the B6----bm 12 combination was only seen in hosts given donor BM. With transfer of donor L3T4+ cells plus host BM, even lightly irradiated recipients died rapidly from hemopoietic failure. Because this syndrome failed to occur in mice given a mixture of donor and host BM, it would appear that L3T4+ cells destroyed host lymphohemopoietic cells by direct cytotoxicity rather than via a bystander effect.

In graft-versus-leukemia (GVL) responses, the cellular subsets and effector mechanisms responsible for cytotoxicity against leukemic cells in vivo remain poorly characterized. A murine model of syngeneic GVL that features CD4(+) and CD8(+) T-cell responses against the MMB3.19 myeloid leukemia cell line has been previously described. MMB3.19 expresses high levels of functional Fas and tumor necrosis factor (TNF) receptors that do not transduce proapoptotic signals. Through the use of perforin- and Fas ligand (FasL)-deficient mice, it was demonstrated that CD4(+) T cells mediate anti-MMB3.19 effects in vivo primarily through the use of FasL and secondarily through perforin mechanisms. Conversely, CD8(+) T cells induce GVL effects primarily through the use of perforin and minimally through FasL mechanisms. Although the in vivo observations of CD8(+) T cells were reflective of their in vitro cytotoxic T lymphocyte (CTL) activity, for CD4(+) T cells, in vitro responses were dominated by the perforin pathway. In addition, the diminished capacity of T cells from perforin- and FasL-deficient mice to lyse MMB3.19 target cells appeared directly related to their deficient cytotoxic functions rather than to defects in activation because these cells were fully capable of mounting proliferative responses to the tumor cells. These findings demonstrate that GVL responses of T-cell subsets can involve preferential use of different cytotoxic mechanisms. In particular, these findings identify a role for both FasL-employing CD4(+) CTLs and the more novel perforin-utilizing CD4(+) T-cell subset in responses against a myeloid leukemia.

Bcl-2 is a potent suppressor of apoptosis, and its overexpression contributes to tumorigenesis in many types of human cancers. To test the possibility of modulating Bcl-2 function as an anticancer strategy, a cell permeable Bcl-2 binding peptide, cell permeable moiety (cpm)-1285, was designed by chemically attaching a fatty acid to a peptide derived from the proapoptotic protein Bad. cpm-1285 entered HL-60 tumor cells, bound Bcl-2 protein, and induced apoptosis in vitro. In contrast, cpm-1285 had little effect on normal human peripheral blood lymphocytes. Furthermore, cpm-1285 had in vivo activity in slowing human myeloid leukemia growth in severe combined immunodeficient mice. These results demonstrate a novel approach for therapeutic intervention of tumor growth in vivo with small molecule inhibitors of Bcl-2.

Allergic reactions are mediated by IgE antibodies bound to high-affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor-associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18-IgE and MOv18-IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18-IgE were greater and of longer duration than those of MOv18-IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.

Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis, is a genetically determined phenotype. In this study, analyses of the cumulative disease frequencies in parental, F1 hybrid, and F2 mice, derived from the EAE-susceptible SJL/J strain and the EAE-resistant B10.S/DvTe strain, confirmed that susceptibility to EAE is not inherited as a simple Mendelian trait. Whole genome scanning, using 150 informative microsatellite markers and a panel of 291 affected and 390 unaffected F2 progeny, revealed significant linkage of EAE susceptibility to marker loci on chromosomes 7 (eae4) and 17, distal to H2 (eae5). Quantitative trait loci for EAE severity, duration, and onset were identified on chromosomes 11 (eae6, and eae7), 2 (eae8), 9 (eae9), and 3 (eae10). While each locus reported in this study is important in susceptibility or disease course, interactions between marker loci were not statistically significant in models of genetic control. One locus, eae7, colocalizes to the same region of chromosome II as Orch3 and Idd4, susceptibility loci in autoimmune orchitis and insulin-dependent diabetes mellitus, respectively. Importantly, eae5 and eae7 are syntenic with human chromosomes 6p21 and 17q22, respectively, two regions of potential significance recently identified in human multiple sclerosis genome scans.

We used an approach of protein surface epitope mapping by synthetic peptides to analyze the surface structure-function relationship of the CD8 protein. Small synthetic peptide mimics of the CD8 DE loop were shown to effectively block CD8 binding to major histocompatibility complex (MHC) class I molecules and possess significant inhibitory activity on in vitro CD8(+) T cell function. These results suggested that the DE loop region of the CD8 protein is an important functional epitope mediating CD8-MHC class I interaction and the activation of CD8(+) T cells, a finding that is consistent with the recently reported crystal structure of the CD8-MHC class I complex. The structural basis for the biological activity of the DE loop peptide was further analyzed in a series of analogs containing alanine substitutions. This study provides support for the concept of bioactive peptide design based on protein surface epitopes and suggests that such an approach may be applicable to other protein-protein complexes, particularly those of immunoglobulin superfamily molecules.

This study was undertaken to distinguish between several mechanisms responsible for graft-vs-host disease (GVHD) protection in anti-CD3epsilonF(ab')2 fragment (Fr)-treated recipients: TCR down-modulation, deletion, failure of expansion, or anergy induction. To quantify alloreactive T cell expansion and function, thoracic duct lymphocytes (TDL) were analyzed. Sixfold fewer donor TDL T cells were recoverable from anti-CD3epsilonF(ab')2 Fr as compared with irrelevant F(ab')2 Fr-treated recipients at the time of peak T cell expansion in vivo. Kinetic analysis revealed that donor T cell expansion was inhibited and not simply delayed by anti-CD3epsilonF(ab')2 Fr. Similar proportions of TDL T cells in irrelevant and anti-CD3epsilonF(ab')2 Fr were undergoing apoptosis. Although TCR modulation was observed, donor TDL T cells had intact anti-host alloresponses as compared with irrelevant F(ab')2 Fr-treated recipients. Because donor CD4+ T cells are primarily responsible for GVHD in this model, an adoptive transfer system was used in which the function and kinetics of expansion of OVA-specific CD4+ TCR transgenic cells could be physically tracked. Relevant Fr severely blunted CD4+ TCR transgenic T cell clonal expansion after OVA administration. Nonviable transgenic and nontransgenic T cells were proportionally similar in OVA-pulsed recipients, regardless of whether relevant or irrelevant F(ab')2 Fr were given. After discontinuing Fr, transgenic T cells were found to have intact in vitro OVA-specific responses. Our current and previous results suggest that reduced donor T cell expansion and T cell depletion both contribute to GVHD protection by anti-CD3epsilonF(ab')2 Fr. These data have implications for designing therapeutic approaches directed toward TCR targeting in humans.

A structure-based designed peptide has been engineered to exhibit the same molecular surface as a portion of the CDR3-like region in domain 1 of the murine CD4 molecule. Earlier in vitro experiments indicated that this analog, known as rD-mPGPtide, inhibited T-cell proliferation in mixed lymphocyte reactions and blocked activation of both normal CD4+ T cells and T-cell lines after T-cell receptor triggering. In addition, rD-mPGPtide proved to be a potent inhibitor in vivo of CD4+ T-cell-mediated experimental allergic encephalomyelitis disease in the SJL mouse model. In this current report, we have evaluated the potential of rD-mPGPtide for suppressing the development of graft-versus-host disease (GVHD) in an irradiated major histocompatibility complex (MHC)-haploidentical murine bone marrow transplantation (BMT) model [(B6 x DBA/2)F1--(B6 x CBA)F1 (950 cGy)]. Our results indicated that early administration of rD-mPGPtide was effective in the inhibition of alloreactive responses of the donor T cells against the host and thus delayed or prevented the onset of GVHD. The median survival time of animals treated with rD-mPGPtide was enhanced as much as four-fold with as little as a single dose of peptide at the time of transplant. Decreased alloreactivity was indicated by phenotypic and functional analysis of positively selected thoracic duct lymphocytes 4 days after transplant and by histopathological examination of skin and gastrointestinal tissue samples 4 weeks later. Therefore, the administration of a CD4-CDR3 peptide is an efficacious approach against the development of GVHD during allogeneic BMT.

We have developed synthetic peptide analogs to analyze novel surface structures of the human CD4 protein potentially involved in T cell activation. Linear and cyclic peptides derived from the FG and CC' loops of the membrane proximal fourth domain of CD4 displayed inhibitory activities in a CD4-dependent immunological assay. These results suggest that the fourth domain of CD4 plays an important role in T cell activation. In addition, we report the synthesis of a highly stable CD4 peptide analog cyclized by the formation of an amide bond between amino and carboxyl termini. Serum stability studies showed that this main-chain cyclic CD4 peptide was highly resistant to proteolytic degradation while the linear and disulfide cyclic peptides were much less stable. The strategy of main chain cyclization of CD4 peptides may represent a promising approach to generate proteolytically stable, orally active immunoregulatory agents.

T cell responses to multiple minor histocompatibility antigens are governed by the complex phenomenon of immunodominance, as demonstrated clearly in the generation of CTL in the C57BL/6By (B6) anti-BALB.B strain combination. Immunodominance has also been found in lethal graft-versus-host disease (GVHD) responses directed to BALB.B minor histocompatibility antigens, after transplantation of B6 T cells and T cell-depleted bone marrow to irradiated (825 cGy) recipients of either the BALB.B or CXB recombinant inbred strains. However, previous results indicated that the hierarchy of immunodominance in GVHD differed from that predicted from the in vitro CTL studies. Lethal GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients, the latter 2 strains expressing immunodominant antigens for CTL generation. A major hypothesis to account for these discordant observations is that GVHD reflected the activity of CD4+, but not CD8+, T cell subsets, in contrast to only the in vitro cytolytic potential of CD8+ T cells. Therefore, the current study was undertaken to assess the GVHD potential of both T cell subsets in the B6--BALB.B, CXBE, CXBI, and CXBJ strain combinations and to analyze the early GVHD responses in the B6--CXBG and CXBK strains. The results indicate that lethal GVHD responses in the B6--BALB.B combination can be mediated by either CD4+ T cells or CD4-dependent CD8+ T cells; a similar observation was made with the B6--CXBI strain combination. Lethal GVHD in the B6--CXBE strain combination is mediated only by CD4-dependent CD8+ T cells, whereas GVHD in the B6--CXBJ combination involves either CD4+ T cells alone or CD4-independent CD8+ T cells. In the B6--CXBG and CXBK recipients, which do not develop lethal GVHD, the early phases of a GVHD response was detected with involvement of both CD4+ and CD8+ T cells. These results indicate that CD8+ T cells are active at some level in all of the strain combinations tested, that CD4+ T cells do not account for the GVHD immunodominant response in the CXBE recipients, and that the failure to obtain extensive clinical disease in the CXBG and CXBK strains is not due to a lack of a graft-versus-host response.

An in vivo model for human melanoma was established with the growth of CR3 and DE5 human melanoma tumor cells following i.v. injection into C.B.-17 severe combined immunodeficient mice depleted of murine natural killer (NK) cells. The ability of human NK cells to mediate antitumor activity in vivo was investigated by evaluating the number of lung nodules and survival of mice given injections of human NK cells i.v. early after injection of CR3 tumor cells. Under these conditions, human NK cells effectively reduced lung nodule counts and prolonged survival when coinjected with interleukin 2 (IL-2). Multiple injections of IL-2 given during the first 16 h post-NK injection did not further enhance the tumor reduction. Significantly increased antitumor activity against CR3 tumor cells in vivo was observed in mice receiving NK cells coinjected with IL-2 and interleukin 12 (IL-12) in comparison to NK cells and IL-2 only. However, coinjection of IL-12 with human NK cells alone did not reduce the tumor burden. These results demonstrate the antitumor activity of human NK cells against human melanoma in severe combined immunodeficient mice and its augmentation by IL-2, alone or in combination with IL-12, suggesting that this model can be used to further investigate the interaction between human NK cells and human tumors.

Lethal graft-versus-host disease (GVHD) can be induced in MHC-matched strain combinations which differ in their expression of multiple minor histocompatibility (H) antigens. It has been shown that CD8+ T cells play an important role in the development of disease directed to the minor H antigens, and that initial indications were that highly purified preparations of these cells were capable of mediating GVHD, without apparent 'help' from mature donor-derived CD4+ T cells. To further strengthen this hypothesis, the current study was undertaken with the B10.BR----CBA strain combination in which irradiated recipient mice were additionally treated with an anti-CD4 monoclonal antibody, as a single or repeated injection, to minimize the presence of either residual host CD4+ cells or recently generated donor-derived CD4+ cells at later stages of disease development. The results indicate that these treatments do not affect the GVHD outcome and that the CD8+ cells are indeed capable of inducing disease independent of CD4+ 'help'. The addition of donor CD4+ T cells in the inoculum, however, does enhance the potential of these CD8+ cells, and is observed with both low and high dosages of CD4+ cells. CD4+ T cells, on their own, have also been observed to cause GVHD directed to minor H antigens in certain strain combinations, and their response has been further characterized in this study. Results indicate that CD4+ cells capable of mediating GVHD in the B10.D2----DBA/2 strain combination can do so over a wide range of recipient irradiation exposures. The transfer of high dosages of CD4+ cells only shortens survival times of the recipients and does not afford any apparent protection phenomenon as previously observed in CD4+ cell mediated anti-class II MHC GVHD. The study also indicates that neither CD4+ nor CD8+ cells responsible for GVHD directed to minor H antigens seem capable of targeting host stem cell elements.

Cryopreserved cell suspensions of freshly excised melanoma metastases from nine patients were injected s.c. into C.B-17 severe combined immunodeficiency (SCID) mice. All 9 tumors grew as s.c. masses and six of nine were successfully transplanted into other SCID mice. Transplant inocula as low as 5 x 10(5) cells resulted in 100% tumor incidence. Moreover, seven of nine tumors metastasized, five from the original s.c. implants and two from transplanted s.c. tumors. Metastases were detected mainly in the lungs but also were found in abdominal viscera (liver, spleen, and pancreas) and thoracic lymph nodes. Flow cytometric analysis showed that expression of a panel of melanoma antigens, melanoma-associated proteoglycan, ganglioside GD3, and ganglioside GD2, was maintained with SCID passage. The original tumor inocula contained a variable percentage of tumor-associated lymphocytes (1-76%). Flow cytometry analysis indicated that these were mainly CD3+ T-cells. However, there was no correlation between the percentage of tumor-associated lymphocytes and the time required for development of a palpable tumor after s.c. injection or the ability to metastasize. These results demonstrate the growth and spontaneous metastasis of fresh human melanoma in SCID mice and suggest that this model could be important for therapeutic and basic biological studies.

Information was sought on the phenotype of lymphoid cells causing lethal graft-vs-host disease (GVHD) in irradiated mice expressing whole or partial H-2 differences. In all strain combinations tested, pretreating donor lymph node (LN) cells with anti-Thy-1 monoclonal antibodies (MAb) plus complement (C) abolished mortality. With GVHD directed to class I H-2 differences, pretreating LN cells with anti-Lyt-2 MAb prevented mortality, whereas MAb specific for Ly-1 or L3T4 cell surface determinants caused severe mortality. These data imply that lethal GVHD directed to class I H-2 differences is mediated by L3T4-, Lyt-2+ cells; this subset of T cells was shown previously to control GVHD directed to multiple minor histocompatibility antigens, i.e., antigens seen in the context of self-class I molecules. With whole H-2 differences, GVHD appeared to be controlled largely but not exclusively by L3T4+, Lyt-2-T cells. This T cell subset was also the predominant cause of GVHD directed to class II differences. With class II incompatibilities, depleting donor cells of L3T4+ T cells, either by pretreatment with anti-L3T4 MAb + C or by fluorescence activated cell sorter selection, greatly reduced but did not completely abolish GVHD. These data might imply that L3T4-, Lyt-2+ cells have some capacity to elicit anti-class II GVHD. A more likely possibility, however, is that the residual GVHD to class II differences observed with Lyt-2+-enriched cells reflected minor contamination with L3T4+ cells.

Mice of the kdkd strain predictably develop a spontaneous tubulointerstitial nephritis after 8 wk of life. In this report we have examined several aspects of the nephritogenic immune response that seemed potentially relevant to the expression of this progressively destructive renal lesion. Of particular interest is that by direct immunofluorescence we were unable to demonstrate the presence of antibodies to determinants in the tubulointerstitium. Serum and kidney eluates from nephritic mice, furthermore, did not stain any renal structures in normal kidney. We did observe, however, that disease could be transferred through kdkd----CBA/Ca bone marrow chimeras, and prevented, in the reverse direction, by CBA/Ca----kdkd chimeras. The development of the interstitial lesion was markedly inhibited by thymectomy with T cell depletion, but disease could not be adoptively transferred with cells or serum from nephritic mice. The interstitial lesions also did not appear in (kdkd X CBA/Ca)F1 hybrids, and the development of disease in kdkd mice could be inhibited by treatment with adoptively transferred T cells from CBA/Ca mice. With these new findings we now hypothesize that susceptibility to the expression of interstitial nephritis in kdkd mice involves the cellular limb of the immune system, and may be related, in part, to alterations in regulatory T cell function.

Detailed information was sought on the capacity of purified Lyt-2+ cells to mediate lethal graft-versus-host disease (GVHD) directed to class I H-2 differences. When B6 Lyt-2+ cells were transferred to irradiated class I-different (B6 x bm 1)F1 mice, three different patterns of lethal GVHD were observed. First, rapid death from hematopoietic failure occurred when Lyt-2+ cells were transferred together with host-type marrow cells; this form of GVHD probably reflected direct destruction of stem cells by Lyt-2+ cytotoxic cells. Second, a pattern of late-onset, chronic GVHD resulting in death only after 4-6 wk occurred when Lyt-2+ cells were supplemented with donor marrow. This syndrome developed in the apparent absence of L3T4+ cells and was observed with either high or low doses of Lyt-2+ cells and with either light or heavy irradiation of the host. Third, an acute form of GVHD resulted when Lyt-2+ cells plus donor marrow cells were supplemented with exogenous help, i.e., by adding small doses of donor L3T4+ cells or injecting the hosts with rIL-2. Although L3T4+ cells potentiated GVHD when injected in small doses, supplementing Lyt-2+ cells with large doses of L3T4+ cells paradoxically led to marked protection; symptoms of GVHD were mild and no deaths occurred.

Intravenous injection of CBA mice with H-2-compatible irradiated B10.BR spleen cells led to a sequence of negative and positive selection of the host T-cell response against the multiple foreign minor histocompatibility antigens (HA) on the injected cells. By 1 d posttransfer, thoracic duct lymphocytes (TDL) of the host had lost the capacity to differentiate in vitro into cytotoxic cells specific for the injected minor HA; spleen and lymph node cells, by contrast, gave normal or enriched responses at this time. By 5 d posttransfer, TDL were hyperresponsive to the injected antigens. Selection with disrupted (sonicated) cells gave similar findings. With injection of either irradiated of disrupted spleen cells, the H-2 haplotype of the minor HA-bearing cells had no apparent effect on the magnitude of selection. By contrast, treatment of spleen cells with glutaraldehyde before injection led to H-2 restriction of selection, i.e., negative selection of the CBA response to B10.BR was marked with injection of glutaraldehyde-treated H-2-compatible B10.BR cells but was minimal with H-2-different B10 or B10.D2 cells. These data are taken to imply that, at least in H-2-incompatible situations, the minor HA-bearing cells must be processed by host cells, i.e., to allow the antigens to become associated with self H-2 determinants. Circumstantial evidence from studies on the specificity of selection induced with glutaraldehyde-treated cells from mice of the B10 recombinant strains suggested that I region-restricted T cells may control the induction of H2K, D-restricted cytotoxic precursor cells.

Systemic inoculation of the interferon (IFN) inducer polyinosinic-polycytidylic polyribonucleotide (poly I:poly C) into CBA/J mice produces a significant decrease in the number of thoracic duct lymphocytes (TDL) collected 6 to 22 hr after injection. The number of TDL is restored to normal levels by 48 to 64 hr. Residual TDL collected during the inhibition period of poly I:poly C treatment showed a phenotypic profile similar to phosphate-buffered saline- (PBS) treated control groups when examined for Lyt-1.1, Lyt-2, Thy-1.2 and Iak surface markers. Interferon is implicated as the mediator of this phenomenon since: 1) Inoculation of exogenous IFN can induce a similar suppression in the number of recoverable TDL. 2) Pretreatment of mice with sheep anti-murine IFN serum can block this effect of poly I:poly C. Similar experiments with vaccinia virus demonstrate that although live virus can partially suppress the TDL output, almost complete suppression is achieved with ultraviolet (UV)-inactivated virus. Vaccinia virus-induced suppression of the number of TDL also appears to be caused by IFN since: 1) UV-inactivated virus induces significantly higher serum levels of IFN in comparison to live virus. 2) The suppression of TDL output by either live or UV-inactivated vaccinia virus can be blocked by pretreatment with anti-murine IFN. These findings suggest that the immunosuppression often associated with viral infections may be at least partially due to a decrease in lymphocyte recirculation mediated by the IFN initially released in response to the virus.

Cytotoxic T lymphocytes (CTL) are able to eliminate P815 (DBA/2) mastocytoma cells growing in cerebrospinal fluid of BALB/c H-2-compatible but minor histocompatibility (H) antigen-different mice and in H-2-incompatible C3H/He mice. We examined the magnitude of the primary CTL response to multiple, minor H antigens and to determinants of the major histocompatibility complex (MHC) by using a direct cytolytic assay and limiting-dilution analysis to estimate CTL frequency. By these criteria, no obvious differences emerged, and the responses appeared comparable at the site of inflammatory process, despite differences in the number of clonal progenitors. Experiments with radiation chimeras showed evidence of a strong cytotoxic T-cell response against P815 cells in [(ddd X bbb)F1----ddd] and (F1----bbd), but not in (F1----bbb) radiation chimeras. Therefore, this cytotoxic T-cell response against minor H antigens obeys the postulated rules for thymic restriction of precursors. Compatibility at the H-2 D-end of the MHC is apparently sufficient to ensure a strong response.

In this report, we have examined the effector T cell repertoire in the spontaneous interstitial nephritis of kdkd mice. Lymph node cells from nephritic kdkd mice are capable of transferring this disease into thymectomized, irradiated, and bone marrow-reconstituted CBA/Ca recipients. CBA/Ca mice do not spontaneously develop interstitial nephritis and are normally resistant to the adoptive transfer of nephritic cells, a resistance that in the short term can be attenuated with low-dose cyclophosphamide. We therefore used delayed-type hypersensitivity responses and direct transfer of immune cells under the renal capsule to characterize nephritogenic effector cells from kdkd donor mice. Lyt-2+, L3T4- T cells from the peripheral lymphoid organs of nephritic kdkd mice, after adoptive transfer into cyclophosphamide-pretreated CBA/Ca recipients, mediate an antigen-specific delayed-type hypersensitivity response to renal tubular basement membrane antigens. These cells are restricted by gene products in H-2Kk; they are also present in nephritic, but not in control kidneys. We have also observed this same phenotypic subpopulation of kdkd lymphocytes mediate a destructive interstitial renal lesion within 7 days of being placed under the kidney capsule of CBA/Ca mice. These findings suggest that T lymphocytes reactive to a parenchymal tubular antigen are of substantial importance in the development of spontaneous interstitial nephritis in kdkd mice.

A psychrophilic marine bacterium was stable in cold seawater or in a solution containing 0.1 M Mg++ and 0.5 M NaCl. In water and in a number of monovalent salts, including up to 3.0 M NaCl, turbidity of suspensions fell and some intracellular material was released into the external medium. The cells first became swollen and pale under phase contrast optics and then disintegrated into amorphous aggregates. Divalent salts alone, some in very low concentrations, could maintain cell structure. Effectiveness was in the order Cu++ > Zn++ > Ni++ > Ca++ > Mn++ > Mg++. The polyamine, spermine, was ineffective. In the presence of 0.1 M Mg++, NaCl and, less effectively, LiCl and RbCl could maintain structure, but KCl, NH4Cl, CsCl, glucose, and sucrose could not. Sodium phosphate, pH 7.0, caused lysis but other sodium salts maintained structure in the presence of Mg++. For growth, Mg++, NaCl, and traces of other ions were required.Phosphatidases attacking cells at 25 °C and higher required salts for maximum activity. The turbidity of water-lysed cells or of envelopes prepared from mechanically broken cells fell at 25 °C and higher; this fall was also dependent on the presence of salts. If cells were lysed in distilled water, or broken mechanically in the presence of salts, more than half their lipid phosphorus and hexosamine was released from material sedimenting after 30 min at 15 000 × g but these materials were not degraded into other substances. The released material seemed bound to smaller particles, since most of it sedimented after 1 hour at 100 000 × g. The lipid composition of smaller and larger particles, and of whole cells, was the same.

The number of cells in the thymus of [k leads to (b X k)F1] radiation (100 rad) chimeras increases approximately 10-fold between 7 and 14 days after reconstitution with bone marrow. At least 50% of the cells in thymus on day 14 are of host origin and respond to virus presented in the context of both H-2k and H-2b when primed in irradiated, virus-infected (b X k)F1 recipients. Strong CTL responses can be generated from thymocytes of donor origin on day 21. All evidence of a significant host thymocyte component has disappeared by day 28. The responsiveness of 14-day thymocytes is not abrogated by pretreatment of the mice used to make the chimeras with anti-thymocyte serum or by using doses of irradiation as high as 1200 rads to eliminate host components.

This report describes a novel system for the immunological detection of immobilized antigen. The detection of herpes simplex virus (HSV) antigen was used as an example. Bacteriophage M13, containing the E. coli lac Z gene, was used as the "reporter" molecule in an immunoassay which is otherwise analogous to the enzyme-linked immunoabsorbant assay (ELISA). Briefly, HSV infected cells were incubated with a mouse monoclonal antibody specific for HSV antigen, followed by rabbit anti-mouse serum and mouse anti-M13 serum. Immune complexes were incubated with viable M13 phage. M13 binding was due to the presence of M13 antibodies, whose presence ultimately depended on the binding of monoclonal antibody to HSV. Phage was recovered by elution in pH = 11. Recovered phage was used to infect E. coli. M13 was quantitated by either plaque assay or by an assay for phage-induced beta-galactosidase activity in appropriate E. coli strains. The amount of M13 recovered was proportional to the number of HSV infected cells probed. Therefore, M13 served as a "bio-amplifiable tag" to antibody, as enzymes do in the ELISA. Since M13 is viable, its signal can be amplified by infection of susceptible bacteria, and the promise for an enormously sensitive immunoassay exists. The sensitivity of the assay described here is compared to the ELISA in the detection of HSV infection cells, as an example of the novel assay's potential. Significantly, the novel assay was more sensitive than the ELISA when samples were tested under identical circumstances. This technique is called the phage-linked immunoabsorbant assay (PHALISA), by analogy to the ELISA.

Following adoptive transfer into irradiated recipients, thymocytes can be induced to respond strongly to vaccinia virus. High levels of cytotoxic T-lymphocyte (CTL) activity may be generated from thymus, but not from spleen, of 3-day-old mice. The capacity of thymocytes to differentiate into effector CTL tends to be lost with age. Some of this loss may reflect positive suppression: a single, low dose of cyclophosphamide allows the reemergence of responsiveness in at least one mouse strain. Thymocytes from [A leads to (A x B)F1] and [(A x B)F1 leads to A] chimeras show the response patterns that would by predicted from previous studies of lymph node and spleen cells. However, thymic function seems to be rapidly lost in the [A leads to (A x B)F1] Chimeras.

The virus-immune responder characteristics of thymocytes, spleen and lymph node (LN) cells from (P1 X P2)F1 leads to P1 radiation chimeras have been examined sequentially at weekly intervals. Adoptively-transferred thymocytes generate strong cytotoxic thymus-derived lymphocyte (CTL) responses from 28 to 100 days after reconstitution with bone marrow, which are almost invariably restricted to recognition of virus presented in the context of P1. This pattern of H-2 restriction is also maintained for spleen and LN cells from the [(H-2kXd)F1 leads to H-2k] and [(H-2kXb)F1 leads to H-2k] combinations but there is random emergence of reactivity to H-2k+virus for peripheral lymphoid cells from [(H-2KkXb)F1 leads to H-2b] chimeras. Treatment of established [(P1 X P2)F1 leads to P1] chimeras with a low dose of cyclophosphamide (Cy) did not lead to the emergence of significant CTL effector function for P2 + virus. Also, administration of a large dose of Cy prior to irradiation of the chimera recipients did not modify the H-2 restriction profile of the chimera, though the level of CTL responsiveness associated with the appropriate H-2 type was apparently enhanced.

We recently reported the biological activity and some of the biochemical characteristics of a factor produced by a human T cell hybrid clone able to block hematopoietic progenitor cell proliferation. This 85-kD protein factor, which we have termed colony-inhibiting lymphokine (CIL), has growth regulatory activity on bone marrow precursors bearing Ia (class II) antigens of either granulocytic-monocytic (CFU-GM) or erythroid lineage (BFU-E and CFU-E). Experiments aimed to investigate the specificity of the inhibitory effect on hematopoietic progenitor cell growth suggested that the expression of HLA-DR surface antigens was required on the target cells. We describe in this communication how DR+ cell lines ceased dividing after a few days of culture in the presence of CIL, whereas DR- cell lines were completely unaffected. The increased DR expression on the ML3 cell surface, mediated by the activity of the gamma interferon (IFN gamma), increases the sensitivity to the growth inhibition factor of the ML3 cell line. To verify the hypothesis that the DR antigens might serve as receptors for the factor, enabling it also to interfere in the immune response, we tested CIL in a mixed lymphocyte reaction (MLR), one of the best known in vitro Ia antigen-dependent T cell-mediated immune responses. CIL is able to block major histocompatibility complex-allogeneic MLR both in human and mouse systems. The data indicate that CIL recognizes a nonpolymorphic structure (presumably on all Ia molecules) presented by stimulator cells of either species, and thereby interferes with specific interactions between stimulator and responder cells. Blocking of the alloantigen stimulation stage is also indicated, since CIL is effective only if added to the culture medium during the first 48 h of the MLR. Finally, mouse monoclonal anti-DR antibodies are able to sharply reduce CIL activity on sensitive DR+ cell lines. CIL may act physiologically as a multifunctional mediator in a complex network that links regulation of bone marrow differentiation and the generation of immune responses.