Siva-1 negatively regulates NF-kappaB activity: effect on T-cell receptor-mediated activation-induced cell death (AICD).

Ligation of TCRs on stimulated T cells leads to activation-induced cell death (AICD) resulting in the downregulation of immune responses, a process essential for T-cell homeostasis. In this study, using transformed T-cell lines such as Jurkat and Do11.10 as cellular models of TCR-mediated AICD, we have demonstrated that the proapoptotic protein Siva-1 is required for TCR-induced apoptosis. Knockdown of Siva-1 rendered T cells specifically resistant to anti-CD3 but not Fas-induced apoptosis. Further, we observed that in Siva-1 knockout Jurkat cells, TCR-mediated activation of the canonical and non-canonical limbs of the NF-kappaB pathway are significantly enhanced as reflected by elevated nuclear levels of p65 and RelB, respectively. In addition, loss of endogenous Siva-1 also resulted in the enhanced expression of NF-kappaB- responsive anti-apoptotic genes such as Bcl-xL and c-FLIP. Interestingly, the c-FLIP(short) was detected only in TCR-ligated Siva-1 knockdown Jurkat cells. These results demonstrate a significant role for endogenous Siva-1, through its inhibitory effect on NF-kappaB activity, in TCR-mediated AICD with implications in peripheral tolerance, T-cell homeostasis and cancer.

Defective surface expression of attractin on T cells in patients with common variable immunodeficiency (CVID).

The proliferative responses of T lymphocytes of a subset of patients with CVID are abnormally low. This may be due to abnormalities in extracellular interactions or signalling defects downstream from membrane-associated receptors. Demonstrating that the T cell receptor signalling was normal, we observed no abnormal pattern of activation-induced tyrosine phosphorylation in cells from CVID patients. Moreover, the addition of exogenous IL-2 increased the low proliferation to mitogens, thus indicating the integrity of the IL-2R signalling apparatus. Attractin is a rapidly expressed T cell activation antigen involved in forming an association between T cells and monocytes. Twenty-four to 48 h after activation by CD3 cross-linking, attractin expression was not up-regulated on the cells of CVID patients despite normal up-regulation of CD25 and CD26. On control cells, however, attractin expression was up-regulated together with CD25 and CD26. The addition of the purified 175-kD attractin was capable of restoring the proliferative response of peripheral blood mononuclear cells following CD3 X-L in the presence of suboptimal concentrations of rIL-2 (10 and 20 U/ml). The effect was dose-dependent with the maximal effect at a concentration of 500 ng/ml, and present at a concentration as low as 50 ng/ml. Due to the likely role of attractin in cell guidance and amplification of the immune response, our results indicate that the lack of up-regulation of the molecule in patients with CVID may in turn affect any further step of productive immune response. Our finding may also imply a potential therapeutic role for this novel molecule.

A biochemical function for attractin in agouti-induced pigmentation and obesity.

Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Attractin: a cub-family protease involved in T cell-monocyte/macrophage interactions.

Attractin is a rapidly upregulated membrane-associated molecule on activated T cells. It is a member of the CUB family of extracellular guidance and development proteins, sharing with them a protease activity similar to that of Dipeptidyl peptidase IV (DPPIV/CD26). Most remarkably, and in sharp contrast to CD26, it is released from the T cell and is presumed to be a major source of a soluble serum-circulating attractin. Genomic sequencing reveals that the soluble form is not a proteolytic product of the membrane form, but is in fact the result of alternative splicing. Recent results prove that the loss of murine membrane attractin results in the mahogany mutation with severe repercussions upon skin pigmentation and control of energy metabolism. In each of these latter instances, there is a strong likelihood that attractin is moderating the interaction of cytokines with their respective receptors. We propose that attractin is performing a similar function in the immune system through capture and proteolytic modification of the N-terminals of several cytokines and chemokines. This regulatory activity allows cells to interact and form immunoregulatory clusters and subsequently aids in downregulating chemokine/cytokine activity once a response has been initiated. These two properties are likely to be affected by the balance of membrane-expressed to soluble attractin.

SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55.

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.

Secreted and membrane attractin result from alternative splicing of the human ATRN gene.

Attractin, initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T lymphocytes, also has been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form. The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' terminal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene. The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.

The mouse mahogany locus encodes a transmembrane form of human attractin.

Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.

Attractin (DPPT-L), a member of the CUB family of cell adhesion and guidance proteins, is secreted by activated human T lymphocytes and modulates immune cell interactions.

Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. We have cloned attractin and find that, as in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common gamma cytokine chain. Except for the latter two domains, the overall structure shares high homology with the Caenorhabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.

Serum high molecular weight dipeptidyl peptidase IV (CD26) is similar to a novel antigen DPPT-L released from activated T cells.

This study demonstrates that the 175-kDa form of dipeptidyl peptidase IV (DPPIV) found in normal human serum is identical with a similarly-sized Ag, DPPT-L, found to be rapidly expressed on the surface of activated T cells. As activation progresses, the expression of DPPT-L reaches a peak on day 3, after which expression falls, whereas expression of the 105-kDa CD26/DPPIV detected by the mAb 1F7 increases, as does the ability to bind adenosine deaminase. The loss of DPPT-L from the surface of activated T cells correlates exactly with the appearance of DPPT-L and DPPIV activity in serum-free tissue culture medium. The release of DPPIV was generally greater from CD4+ cells than from CD8+ T cells, and within the CD4+ subset, the CD45RO+ subset was the major source, which correlated with surface expression before culture. We show that the DPPIV released from activated T cells is antigenically, biochemically, and enzymatically similar to DPPIV circulating in the serum and is distinct from the DPPIV activity of 105-kDa CD26. The T cell-released DPPIV is able to function as a costimulating molecule for the response to the recall Ag, tetanus toxoid, at levels similar to those at which recombinant soluble CD26 and serum DPPIV exhibit costimulatory function, suggesting that the released DPPIV may serve an important immunoregulatory function in vivo, both locally and within the systemic circulation.

A novel form of dipeptidylpeptidase IV found in human serum. Isolation, characterization, and comparison with T lymphocyte membrane dipeptidylpeptidase IV (CD26).

Human CD26, a Type II membrane glycoprotein with intrinsic dipeptidylpeptidase IV (DPPIV) activity and ability to bind adenosine deaminase type I (ADA-1), is expressed on epithelial cells constitutively, but on T lymphocytes its expression is regulated. A soluble form of CD26/DPPIV has been described in plasma and related to immunological status, but it has been defined by the presence of DPPIV activity rather than by isolation. Using nondenaturing chromatographic techniques followed by nondenaturing native preparative electrophoresis, we obtained a homogeneous preparation of soluble serum DPPIV and compared it with a recombinant soluble CD26/DPPIV (rsCD26). We show that serum DPPIV is a monomer of 175 kDa in contrast to rsCD26 of 105-110 kDa, that it exists as a trimer, and that it is probably a serine proteinase. Deglycosylation removed N-linked sugar from both serum DPPIV and rsCD26; no O-linked glycosylation was observed, revealing a protein core of 130 kDa for serum DPPIV. The large serum form expresses functional DPPIV activity with substrate and inhibitor specificities and pH activity profile similar to those of rsCD26. Epitope analysis showed that monoclonal antibodies against five epitopes expressed by rsCD26 also bound, but more weakly, with serum DPPIV. Analysis of peptides after limiting proteolysis and N-terminal sequences reveals no homology with rsCD26 but some identity with other peptidases. Unlike rsCD26, the serum form does not bind ADA-1 and has no ADA-1 already associated with it. Similarly to rsCD26, serum DPPIV is a potent T cell costimulator. We conclude that the serum form of DPPIV is unique and is not a breakdown product of membrane CD26. The conservation of DPPIV activity and five epitopes specific to rsCD26 suggest, however, a significant structural similarity.

Role of CD26/dipeptidyl peptidase IV in human immunodeficiency virus type 1 infection and apoptosis.

To examine the role of CD26/dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) in infection by human immunodeficiency virus type 1 (HIV-1), we utilized CD26 cDNA-transfected Jurkat T-cell lines. Both CD26- parental Jurkat cells and mutant CD26+ (DPPIV-) transfected Jurkat cells were readily infected with HIV-1, whereas wild-type CD26+ (DPPIV+) transfected Jurkat cells were more resistant to HIV-1 infection. Our results suggest that CD26 is not essential for HIV-1 infectivity as suggested by others but that DPPIV enzyme activity may decrease the efficiency of HIV-1 infection. Of great interest, we found that mutant CD26+ (DPPIV-) transfectants and CD26- parental Jurkat cells strongly expressed CD95 (Fas/Apo-1) and were more sensitive than wild-type CD26+ (DPPIV+) transfectants to the induction of apoptosis by anti-CD95 monoclonal antibody. These results suggest that CD26 may play a role in HIV-1-associated loss of -CD4+ cells through the process of programmed cell death.

Enhancement of antigen-induced T-cell proliferation by soluble CD26/dipeptidyl peptidase IV.

The addition of a soluble recombinant CD26 (sCD26) enhanced proliferation of peripheral blood lymphocytes induced by the recall antigen tetanus toxoid. sCD26 itself did not provide a mitogenic signal and did not augment the proliferative response of T cells to other mitogenic stimuli such as phytohemagglutinin and anti-CD3. Dipeptidyl peptidase IV-negative sCD26 did not have this enhancement effect, implying a requirement for enzyme activity. It was found that there exists a large variation in the levels of human plasma sCD26/dipeptidyl peptidase IV in vivo which may regulate T-cell activity. Peripheral blood lymphocytes from individuals whose plasma sCD26 was high and responded strongly to tetanus toxoid stimulation were insensitive to the enhancing effects of exogenously added sCD26. This suggests that plasma sCD26 had modulated the responsiveness of T cells of these individuals in vivo and that the endogenous plasma sCD26 regulates immune responses by allowing antigen-specific T cells to exert a maximal response to their specific antigen.

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

Depletion of the helper/inducer (memory) T cell subset using a bispecific antibody-toxin conjugate directed against CD4 and CD29.

We have developed a bispecific antibody that recognizes the CD4 and CD29 antigens simultaneously and that was examined for its ability to target CD4+CD29bright T cells. The premise for using bispecific antibody-toxin conjugates was that monovalent binding to one antigen would not result in internalization while binding through both antigens would predispose toward endocytosis and delivery of the toxin to the cell interior. In this study, we show that the bispecific antibody binds monovalently to CD4 and CD29 and bivalently to both antigens. Both monovalent and bivalent binding rendered the target cells sensitive to complement-mediated lysis, demonstrating that this effector modality cannot take advantage of the dual specificity of the antibody. Bivalent binding, however, allowed modulation of more than 60% of the bound antibody off the surface of CD4+CD29bright cells, which we further exploited to deliver a toxin moiety preferentially to CD4+CD29bright cells. Immunoconjugates incorporating blocked ricin (a ricin holotoxin that has its intrinsic galactose-binding sites blocked by chemically linked affinity ligands) preferentially killed CD4+CD29bright cells in vitro by a factor of 25 in comparison with killing of total CD4+ cells in functional assays. Assays on resting PBL demonstrated a similar specificity of the bispecific immunotoxin for CD4+CD29bright cells with a concomitant relative survival of CD4+CD29dim (CD45RA+). We demonstrated that the potency of the immunotoxin is not only a function of its affinity but also of its propensity to internalize, and that this property is influenced by the degree of bivalent binding. These results open up the possibility of engineering bispecific antibody toxin conjugates for use as therapeutic immunotoxins for selective removal of restricted T cell subsets.

Targeting of an activated T-cell subset using a bispecific antibody-toxin conjugate directed against CD4 and CD26.

We have developed a bispecific antibody that recognizes the CD4 and CD26 antigens simultaneously and that was examined for its ability to target CD4+CD26+T cells. These latter cells constitute the activated component of the CD4+ CD29highCD45RO+ memory T-cell subset that provides help for B-cell Ig synthesis and help for responses against recall antigens. The purified bispecific antibody exhibited an estimated dissociation constant (kd) of 2.4 x 10(-9) mol/L, on comparison with 1.1 x 10(-9) mol/L for anti-CD26, and 1.6 x 10(-10) mol/L for anti-CD4. Surface plasmon resonance was used to show the bifunctional capacity of the antibody. On binding 125I-bispecific antibody to phytohemagglutinin (PHA)-activated T cells, 54.4% of the bound antibody was internalized. This was the result of bispecific binding, because monovalent fragments of anti-CD4 and anti-CD26 were not able to modulate antigen or induce internalization using both a fluorescent assay and an 125I-internalization assay. The ability of the bispecific antibody to be internalized was used to deliver a toxin, blocked ricin, specifically to cells that are CD4+CD26+. The inability of monovalent fragments to be internalized formed the basis for our hypothesis that monovalent binding by the bispecific immunotoxin would not result in internalization. Against resting E+ T cells, the bispecific immunotoxin developed a minimal effect. On preactivating the same cells, using phorbol myristate acetate (PMA)/ionomycin on concanavalin A (ConA) or especially PHA, levels of CD26 were upregulated and the immunotoxin effectively inhibited the ability to provide help for B-cell Ig synthesis while leaving intact the CD4-CD26+ and CD4+CD26- populations; an effect observed both functionally and by phenotype. The bispecific antibody proved to be most effective at inhibiting a heterologous mixed leukocyte reaction. We propose that this reagent may form the basis for the rational design of toxins designed to modulate activated T cells from, or directed against, tissue grafts.

Regulation of CD4-p56lck-associated phosphatidylinositol 3-kinase (PI 3-kinase) and phosphatidylinositol 4-kinase (PI 4-kinase).

CD4 serves as a receptor for MHC class II antigens and as a receptor for the human immunodeficiency virus (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. Although anti-CD4 crosslinking may increase lck activity, the effects of HIV-1 gp120 have been controversial. Activated protein-tyrosine kinases are known to associate with certain intracellular proteins possessing src-homology regions (SH-2 domains) such as phosphatidylinositol 3-kinase (PI 3-kinase). In this paper, we demonstrate that the CD4:p56lck complex associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis of the reaction products demonstrated the presence of phosphatidylinositol 3-phosphate (PI 3-P) and phosphatidylinositol 4-phosphate (PI 4-P), thus indicating that PI 3 and PI 4 kinases associate with CD4-p56lck. The p85 subunit of PI 3-kinase was also detected in anti-CD4 immunoprecipitates by immunoblotting with anti-p85 antiserum. Significantly, p56lck binding to CD4 appears to be necessary for the detection of lipid kinase activity associated with p56lck. Also, anti-HIV gp120 and anti-CD4 crosslinking induced a 10-15-fold increase in levels of both PI 3- and PI 4-kinase activity in anti-CD4 precipitates. Stimulation of CD4-p56lck-linked PI kinases by crosslinked HIV-1 gp120 may play a role in HIV-1-induced immune defects.

The reaction against autologous lymphoblasts as an indicator of lymphocyte hyperreactivity in rheumatoid arthritis.

We have previously found that there is a considerable variation in the responses of lymphocytes of normal individuals to autologous pokeweed mitogen (PWM)-induced lymphoblasts (PWM.AMLR) under completely autologous conditions. Having proposed that the reaction might measure an innate sensitivity for B cell hyperreactivity, we measured the PWM.AMLR of a normal group (8/18 positives) and a group of patients with rheumatoid arthritis (RA; 24/25 positives). Responses of the RA group were in general an order of magnitude greater than those of normal controls that generated positive responses, and the responding cells could clearly be shown to be both CD4-bearing and CD8-bearing T cells. T cell-independent B cell mitogen-induced (staphylococcus A) lymphoblasts derived from RA patients were capable of strongly stimulating autologous responder cells, while similarly treated cells of normal controls induced small responses. The staphylococcus A responses were smaller in general than those induced by PWM-induced lymphoblasts. These results support previous results that the degree of activation of both T cells and B cells determines the size of response in the autologous PWM.AMLR and that the PWM.AMLR may be used to determine differing degrees of the in vivo priming of these cells and their relation to clinical lymphocyte hyperreactivity.

Use of an autologous reaction in vitro to assess contributions of T and B lymphocytes to immune hyperreactivity of atopics.

The in-vitro proliferation reaction of peripheral blood lymphocytes (measured by [3H]thymidine incorporation) to autologous pokeweed mitogen (PWM)-induced lymphoblasts (PWM-lymphoblast-stimulated autologous mixed leucocyte reaction, PWM.AMLR) was used as a measure of immune hyperreactivity for comparison of atopic with non-atopic individuals. Accordingly, 10/24 non-atopics responded in the PWM.AMLR, and 19/19 atopics reacting to inhaled allergens responded. Autologous stimulation was associated with release of mitogenic factors from the PWM-activated stimulating cells (2/15 non-atopics, 9/15 atopics). For non-atopics, stimulation delivered by staphylococcus A (SAC)-activated cells was similar to that delivered by PWM-induced cells, while in atopics, the SAC.AMLR was never more than 50% of the PWM.AMLR, indicating a possible T cell component. Separation by panning of the stimulation cells into lymphocyte subsets supported the notion that stimulation involved a cooperation between B and T4+ T cells. It is proposed that a positive PWM.AMLR is dependent upon an initial B cell activation followed by the PWM stimulus dependent upon a previous T cell activation, where atopics have more lymphocytes in an activated state than healthy non-atopics. Such a baseline priming may contribute to an innate sensitivity of atopics to environmental allergens.

Observations on the contributions of environmental restraints and innate stem cell ability to hematopoietic regeneration.

A competitive repopulation assay utilizing chromosome markers was used to assay the reconstituting potential of hematopoietic populations. The test populations consisted of tibial murine marrow locally irradiated with doses ranging from 1.5 Gy to 8.5 Gy and of marrow generated from either murine splenic or marrow stem cells. The purpose of this assay was to assess the innate proliferative potential and microenvironmental influences on the ability to repopulate. Regardless of origin, spleen repopulating ability consistently agreed with spleen colony-forming unit (CFU-s) content. Doses of radiation from 5 Gy to 8.5 Gy diminished, by a factor of 2, the ability to repopulate marrow despite maintenance of CFU-s levels. Marrow generated from splenic stem cells had one-fifth the repopulating ability of marrow derived from marrow stem cells, even though CFU-s levels were equivalent. The results imply that the splenic environment can only maintain stem cells at the level of the CFU-s, even if the stem cells were originally of higher quality, and that their original potential cannot be regained in a marrow environment. Nevertheless, the marrow can maintain more primitive stem cells, but this reserve is drained to support CFU-s levels.