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T cell inhibitory receptors upregulate in parallel with activating receptors to prevent overwhelming immune response. Excessive or persistent inhibitory receptor expression on T cells, however, has been associated with several pathologic states, including chronic viral infection and malignancy. Programmed death-1 (PD-1) is among the most well studied inhibitory receptors, and PD-1 inhibition has resulted in regression of solid tumor malignancies and improved control of HIV infection. To examine the role of PD-1 in T cell function post allotransplant we examined PD-1 expression on total and CMV antigen specific T cells on samples collected from 43 patients between 55 and 85 days post cord blood transplant (CBT) (n=13),matched unrelated donor transplant (MURD) (n=12), matched related donor transplant (MRD) (n=18) and compared results to normal adult peripheral blood controls (PBMCs) (n=22), and cord blood controls (CB) (n=11). PD-1 expression was significantly upregulated (p We next assessed PD-1 expression on CMV specific CD8+ T cells from HLA A2 patients using a CMVpp65 peptide (NLVPMVATV) specific A2 tetramer. Among 15 CMV positive patients assessed between 55 and 85 days post-transplant, tetramer+, CD8+ T cells were detectable in 10 patients. In patients who effectively controlled CMV clinically, defined as no reactivation (n=12) or reactivation controlled within 1 week of preemptive therapy (n=2), tetramer+, CD8+ T cell populations consisted primarily of EM/EMTD cells (28.9-98.2% of cells, median 75%, n=8) and PD-1 expression was comparable, though modestly increased, compared to total CD8+ T cell populations. In 2 patients not controlling CMV, tetramer+, CD8+ T cells had a greater proportion of naïve/CM phenotype (45.7-54.6% of cells, median 50%, n=2) and expressed markedly higher PD-1 levels than total CD8+T cells. To investigate the potential for PD-1 pathway blockade to enhance CMV responses, we performed stimulations with and without PD-L1 antibody blockade on 17 patient samples collected between 55 and 85 days post-transplant. Eleven patients demonstrated T cell proliferation in response to CMV. For nine of 11 patients, proliferative responses in CD4+ T cells were enhanced by PD-L1 antibody blockade and for eight of 11 patients proliferative responses in CD8+ T cells were enhanced by PD-L1 antibody blockade. Our observations suggest different patterns of T cell immune reconstitution depending on donor source. Similar patterns of immune reconstitution were demonstrated in the case of CMV specific responses. Our data suggests that PD-1 interactions might be manipulated to affect immune responses following transplantation. Further investigation of PD-1 and its interactions with other inhibitory receptors as well as the potential to manipulate PD-1 to enhance GVT and abrogate GVHD are ongoing. Disclosures: No relevant conflicts of interest to declare.

Aim Virtual crossmatches (VXM) are used by transplant programs to evaluate compatibility with potential donors using a recipient’s HLA antibody profile and donor HLA typing. The aim of this study was to determine if the MFIs of single antigen donor specific antibodies (DSA) could be used to predict flow cytometric crossmatches (XM). Methods Patient samples were screened for antibodies to mismatched donor antigens using Luminex single antigen (LSA) beads. The LSA bead MFI values were then correlated with the XM value. Only single antigen DSA was used for this study. Patients with anti-Cw or anti-DP antibodies and MFI values on beads known to give false positive results were also removed. Results The mean LSA bead MFI values of the recipients correlated with their XM values (r2 = 0.81 for class I DSA; r2 = 0.75 for class II DSA). A significant difference between the DSA MFI values in patients whose XM was weak versus strong was found for both class I and class II antibodies. A cutoff MFI value of ⩾2000 predicted 100% of the moderate and strong XMs for both class I and class II antibodies. A prospective XM would be recommended for LSA bead MFI values from 1000-1999 (grey zone). [Fig. 1] Conclusions A patient’s DSA profile can be used for a VXM and its predictive power is better when the LSA MFI is higher. Single antigen DSA MFI values can be used to predict moderate and strong XM. Further analysis of multiple DSA with VXM needs to be performed.

Aim Disease susceptibility for type 1 diabetes (T1D) is strongly associated with the inheritance of specific HLA alleles. However, weak HLA associations may not be detected because many alleles are relatively rare and disparate alleles that have similar peptide-binding sites (shared epitopes) are not included. Methods We developed an R-based Epitope Analysis Program that can detect every possible epitope of 1-4 amino acids in the peptide-binding groove. The amino acids did not have to be contiguous. The number of individuals carrying at least one copy of the epitope was compared with the number of people not carrying the epitope. Results Two class I epitopes were most associated with T1D susceptibility: C 67 in the HLA-B locus ( p = 1.7 x 10 −6 , OR = 1.93) and IYH 95,113,184 in the HLA-C locus ( p = 9.2 x 10 −5 , OR = 1.84). Stronger associations were found with class II, including DRB1 H 13 ( p = 4.2 x 10 −57 ) and K 71 ( p = 6.0 x 10 −36 , OR = 3.59), DQA1 S 26 ( p = 1.0 x 10 −80 , OR = 7.15) and DQB1 A 57 ( p = 1.3 x 10 −87 , OR = 12.1). DRB1 R 13 ( p = 3.9 x 10 −34 , OR = 0.15), DRB1 A 71 ( p = 3.5 x 10 −36 , OR = 0.11), DQA1 Y 80 ( p = 2.3 x 10 −51 , OR = 0.22) and DQB1 D 57 ( p = 7.1 x 10 −97 , OR = 0.11) were strongly associated with resistance. A dominant resistance phenotype was seen for people bearing a protective HLA epitope, even in the presence of a susceptibility epitope. In addition, early disease onset was associated with significantly greater numbers of susceptibility epitopes and significantly fewer resistance epitopes ( p & Conclusions Identifying HLA epitopes associated with T1D may lead to a better understanding of genetic susceptibility and help predict peptides that trigger autoimmunity. Download full-size image

Aim Rheumatoid Arthritis (RA) is a chronic inflammatory disorder affecting 1% of the world population and results in the destruction of bone and joints. A two amino acid shared epitope LA 67,74 in HLA-DRB1 is highly associated with RA and is positively correlated with the level of anti-citrullinated protein antibodies. In contrast, a single amino acid shared epitope, D 70 in HLA-DRB1, is associated with resistance in a resistance dominance fashion. The HLA function in autoimmunity is unclear, but it is possible that citrullination of joint peptides increases their affinity to susceptible HLA molecules. In this study, we utilize a novel method for measuring HLA:peptide binding to answer this question. Methods We measured binding of biotinylated citrullinated and native vimentin peptides to HLA molecules attached to Luminex beads; binding was detected with PE-streptavidin. Results Of the 91 HLA class II molecules, citrullinated vimentin bound over background on 88 of them. Citrullinated vimentin bound significantly more strongly than native vimentin to alleles with LA 67,74 (p = 0.0003). We observed no difference in binding between citrullinated and native vimentin peptides to alleles with D 70 [Fig. 1]. Conclusions Citrullination of vimentin increased the peptide affinity for DRB1*LA 67,74 but not DRB1*D 70 . While native vimentin binds effectively to DRB1*D 70 , it does not bind well to DRB1*LA 67,74 . We also show peptides bind class II HLA (including alleles not associated with RA susceptibility or resistance) over a broad range, from very strong binding (MFI > 5000) to weaker binding (MFI ∼ 100). This demonstrates that HLA epitopes may mediate autoimmunity by preferentially binding autoantigens. Download full-size image

Hydroquinone (HQ) is a benzene derivative that is found in large quantities in cigarette tar as a result of the pyrolysis of tobacco flavinoids. HQ is a potent inhibitor of T cell proliferation, causing an immediate and complete cessation of DNA synthesis in IL-2-dependent human T lymphoblasts and Jurkat T cells without loss of cell viability. Previous studies from our laboratory demonstrated that the antiproliferative effects of HQ could be partially reversed by the addition of deoxyribonucleosides, but not by the corresponding ribonucleosides, suggesting that HQ might inhibit ribonucleotide reductase. In the present study, the molecular mechanism behind this observation was further investigated. Jurkat T cells were stably transfected with a pMEP4 expression vector containing the gene for the M2 subunit of ribonucleotide reductase under transcriptional control of the human metallothionein IIA promoter. M2-transfected Jurkat T cells exhibited a greater than three-fold increase in resistance to HQ compared to untransfected cells or cells transfected with the M2 gene in the reverse orientation. HQ resistance was associated with an increased level of M2 protein detected by Western blot. These results suggest that the benzene derivative inhibits lymphocyte proliferation by inhibiting ribonucleotide reductase.

Cigarette smoking has been shown to cause profound suppression of T-cell responses in the lungs, but the mechanism by which this phenomenon occurs is not known. We have shown that 10 microM p-benzoquinone (p-BQ), a thiol-reactive benzene derivative found in cigarette tar, inhibits mitogen-induced IL-2 production by human peripheral blood mononuclear cells by 76 +/- 7% without affecting lymphocyte/macrophage agglutination or blast transformation. The effect of p-BQ appeared to be specific for IL-2 production, since de novo induction of the IL-2 receptor alpha-chain (CD25) and ICAM-1 (CD54) and upregulation of LFA-1 alpha/beta (CD11a and CD18) were unaffected. In contrast, N-ethylmaleimide (NEM), another alpha,beta-unsaturated diketone with thiol-reactive properties similar to those of p-BQ, inhibited all of these Con A-induced activation events. These results suggest that p-BQ inhibits T-cell mitogenesis by blocking a thiol-dependent event that controls IL-2 production but not other T-cell activation events.

Aim To demonstrate that 1) DTT is effective in removing interfering factors, such as the C1 component of complement, in the Single Antigen (SAB) and C1q assays, 2) when serum is treated with DTT there is good correlation between SAB and C1q, and 3) DTT treatment of sera in the C1q assay provides a valid internal positive control MFI value. Methods: DTT treatment 10 μ l of 0.05M DTT working solution is mixed with 90 μ l of patient serum for a final DTT concentration 0.005M. SAB assay 10 sera which had demonstrated interference by C1 following dilution studies and 6 sera which had demonstrated unexpected positive flow cytometry crossmatch results were treated with DTT and SAB testing was repeated. C1q assay 4 of the same sera that were used to validate the use of DTT in the SAB assay were used to validate the use of DTT in the C1q assay because these sera are known to contain antibodies capable of binding complement. All 4 sera were run in the C1q assay according to the manufacturer’s instructions (with the exception of heat inactivation) and compared to results of IgG subclass testing. Results: SAB assay All 10 sera showed that interference from C1 was eliminated. The 6 sera whose crossmatch results were much stronger than expected, or unexpectedly positive, showed increases in DSA MFI values after DTT treatment. SAB MFI values of the DSA following DTT treatment correlated well with the MFI values of the crossmatches. C1q assay Results of C1q testing without DTT treatment did not correlate well with what was expected based on the results of the SAB DTT validation. If the C1q test detects complement binding antibodies, it should show reactivity with the same antibody specificities that were inhibited by C1 in the SAB assay. Repeat testing of the 4 sera using PE-conjugated monoclonal antibodies to the 4 IgG subclasses confirmed that complement binding antibody was present. The antibodies were predominantly IgG1 and they were binding to the same specificities that had been blocked in the SAB assay due to C1 interference. A modified C1q procedure in which DTT treatment could be done without interfering with the C1q reagent was developed. Results of the modified procedure correlate very well with results of both the IgG subclass testing and the SAB DTT validation. It was also discovered that DTT treatment of serum in the C1q assay results in valid positive control bead MFI values.

Hydroquinone (HQ) is a major metabolite of benzene and is present in large quantities in cigarette tar as a result of the combustion of tobacco leaf pigments. We hypothesize that the immunosuppressive effects of cigarette smoking are due, in part, to the deposition of large quantities of HQ in the lungs. Exposure of primary human T lymphoblasts (HTL) in vitro to 50 μ m HQ blocked IL-2-dependent proliferation by >90% with no loss in viability. Inhibition of DNA synthesis was observed immediately after the addition of HQ to the cells. However, this effect could be reversed up to 6 hr later by simply washing the cells and reculturing them in the absence of HQ. HQ did not significantly alter intracellular glutathione levels up to 24 hr later, and the presence of 50 μ m 2-mercaptoethanol or 500 μ m dithiothreitol during the treatment did not prevent inhibition of DNA synthesis. HQ did not block binding of 125 I-IL-2 to the cells, but inhibited the IL-2-dependent progression of HTL through S phase of the cell cycle. These observations demonstrate that HQ, in concentrations comparable to those found in cigarette tar, is a potent inhibitor of IL-2-dependent T cell proliferation and may therefore help to explain the potent immunosuppressive effects of cigarette smoke on lung T lymphocytes.

The article highlighted in this issue is "The Activity of NF-kappaB in Swiss 3T3 Cells Exposed to Aqueous Extracts of Cigarette Smoke Is Dependent on Thioredoxin," by Stephan Gebel and Thomas Müller (pp. 75-81).

Abstract 3772 Genetic modification of autologous hematopoietic stem cells (HSC) has the potential for effective treatment of a wide variety of inherited blood disorders. However, HSC gene therapy has shown limited clinical efficacy (with notable exceptions), in part because of the small proportion of engrafted genetically corrected HSCs. The use of drug-resistance genes to enable selection for transduced HSCs has been explored, but with limited success. Previous studies from our laboratory have indicated that murine HSC can be selected with 6-Thioguanine (6TG), a relatively non-toxic drug used in the treatment of leukemias, after knocking down the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT), an enzyme that metabolizes 6TG to its active state. We sought to determine if these findings can be translated to human hematopoietic cells. In the present study, we transduced human myeloid (Molm13, MV4-11) and lymphoid cell lines (Reh) with lentiviral vectors expressing shRNA constructs targeting HPRT or a non-targeted control sequence (Ctrl). Two of the most promising constructs directed against HPRT (491 and 50) were studied in more detail to determine which is most effective. Cells were selected in puromycin and cell lysates analyzed for HPRT gene expression. Reverse-transcription, real-time PCR (RT qPCR) and western blotting demonstrated that construct 491 was most efficient in knocking down HPRT in human hematopoietic cell lines compared to construct 50 (and Ctrl). To determine whether knockdown of HPRT provided resistance to 6TG, cells were cultured in the absence or presence of different doses of 6TG and live cell concentrations were determined. While Ctrl transduced cells decreased in a dose dependent manner after 72h of 6TG treatment, cells transduced with constructs 491 and 50 were relatively resistant to 6TG. IC50 values for construct 491 were significantly higher (114μM for Molm13 and 46μM for Reh cell lines) than construct 50 (1μM for Molm13 and 10μM for Reh) in comparison to control transduced cells (0.4μM for Molm13 and 3.5μM for Reh). We assessed cell death in human hematopoietic cell lines by annexin V staining after exposure to 6TG at 48 and 72h. As expected, control transduced cells died of apoptosis upon 6TG treatment, while 491 and 50 transduced cells were resistant. Furthermore, 491 transduced cells were more resistant to apoptosis than 50 transduced cells. Based on these results, construct 491 was used to transduce human CD34+ progenitor cells isolated from umbilical cord blood along with control shRNA. Transduction efficiency varied from 25–35% as determined by %GFP expression by flow cytometry. Sorted GFP+ cells showed reduced expression of HPRT in 491 transduced cells compared to controls, as measured by RT qPCR. Similar to the effects in cell lines, in vitro proliferation of control transduced CD34+ cells diminished in response to increasing 6TG concentrations. There was an increase in the percentage of GFP+ cells in 6TG treated 491 transduced cells compared to untreated controls in a dose dependent fashion, indicating a selective advantage conferred to 491 transduced cells in the presence of 6TG. Importantly, 491 transduced cells continued to proliferate despite treatment with 6TG. Like 6TG, cisplatin requires mismatch repair (MMR) for cytotoxicity. To determine if HPRT knockdown had off-target effects impairing MMR, transduced cells were also treated with cisplatin. Both control and 491 transduced cells stopped proliferating in the presence of cisplatin indicating that MMR remained intact. These data indicate that human hematopoietic progenitor cells can be selected in vitro by knock-down of HPRT and treatment with 6TG. Xenografts of Ctrl and 491 transduced human CD34+ cord blood cells have been generated and are being treated with 6TG to determine if human cells can be selected with 6TG in vivo. Disclosures: Off Label Use: Off label use of 6-thioguanine will be suggested.

Although rheumatoid arthritis has long been associated with HLA shared epitopes, a systematic search for epitopes has never been reported. We have developed a computer program that can analyze large HLA data sets and detect all shared epitopes comprising from one to five amino acids. We analyzed high resolution data from the International Histocompatibility Working Group, which included 639 subjects with rheumatoid arthritis and 429 controls, for all combinations of up to five amino acids among HLA-DRB1 positions 9-86. We compared this analysis with a traditional allele analysis and the prevalence of the QRAA epitope. Statistical significance was determined using a Chi-square analysis with a Bonferroni correction. The allele analysis identified three HLA alleles that were positively associated with RA, DRB1*0401 (p=2x10-9), *0404 (p=0.006) and *0405 (p=3x10-5) and five alleles that were resistant, DRB1*0701 (p=0.007), *0801 (p=0.037), *1101 (p=0.037), *1301 (p=0.04) and *1302 (p=0.004). DRB1*0101 (p=0.24) and *0102 (p=0.421) were not associated with RA in this data set. The single amino acid analysis identified V11 (p=10-16), H13 (p=10-16), Y37 (p=10-5), L67 (p=3x10-9), Q70 (p=0.0001) and A74 (p=0.0007) as RA-susceptibility markers. In addition, S11 (p=4x10-7), S13 (p=2x10-6), I67 (p=3x10-7) and D70 (p=5x10-9) were RA-resistance markers. The combination of these positions yielded a VHYLA11,13,37,67,74 epitope that was more strongly associated with RA (p=4x10-22) than QRAA70,71,73,74 (p=9x10-10) or QKAA70,71,73,74 (p=3x10-10). VHYLA is associated with DRB1*0401,*0404,*0405,*0408, *0409 and *0410, but not with DRB1*01 alleles, consistent with the allele analysis in this data set.

The total nucleated cell count (TNC) is a major factor in the acceptance or rejection of an umbilical cord blood (UCB) unit. Yet it is stem cell potency that will decide whether a UCB unit has the capability to engraft and repopulate the patient after transplantation. The colony-forming cell (CFC) assay has been employed in a retrospective manner in order to document the possibility of growth potential of stem cell products destined for transplantation. Although some centers count and differentiate colonies, many use the assay to document either growth or no-growth because the results are difficult to interpret and provide little predictive value. Subjectivity and lack of an external standard to which the assay can be calibrated and validated means that the CFC assay can neither be employed as a reliable and reproducible stem cell potency assay, nor can it be used to define release criteria of UCB products for transplantation. A cell potency and release assay is required by standards and regulatory organizations. Viability and CD34 may be important for engraftment, but cannot measure stem cell potency. In a recent article in Transfusion (48:620, 2008), Reems et al describe a novel instrument-based, ATP bioluminescence proliferation assay used for UCB and compared at two geographical locations. Results demonstrated a correlation for the assay between the locations with a correlation coefficient of R=0.94 (p

treated according to this new protocol (p 0.0003 versus the occurrence of BOS during the previous protocol). Survival, from four months after transplantation, also significantly improved (p 0.05). Conclusions: We conclude that EBV-DNA guided pre-emptive reduction of immunosuppression is a safe procedure after lung transplantation and may not only reduce the incidence of PTLD but also improve the BOS free survival.

To evaluate the efficacy, safety, and cost of preemptive ganciclovir therapy in cytomegalovirus (CMV)-seropositive renal transplant recipients treated with antilymphocyte antibody (ALA) preparations.A prospective, randomized trial at a 650-bed tertiary medical center hospital.Forty consecutive CMV-seropositive renal allograft recipients who underwent transplantation between January 1992 and January 1994 and were treated with ALA for induction immunosuppression or acute rejection therapy.The incidence and severity of CMV disease, length of hospitalization, and patient and allograft survival.Cytomegalovirus infection prophylaxis by use of intravenous ganciclovir during ALA therapy was administered to 22 patients (group 1) and the results were compared with those obtained in 18 control patients who did not receive prophylaxis for CMV disease (group 2).Preemptive ganciclovir therapy significantly reduced the incidence of CMV disease (P.05) in CMV-seropositive renal transplant patients who were treated with ALA and was well tolerated. In addition, the cost of prophylactic therapy was offset by the decreased length of hospitalization observed in patients in group 1.Preemptive ganciclovir therapy provides a cost-effective approach toward significantly improving the outcome of renal transplantation in CMV-seropositive patients treated with ALA.

To compare the efficacy, safety, and cost of prophylactic low-dose ganciclovir with that of immunoglobulin in renal transplant recipients at risk for primary cytomegalovirus (CMV) disease.A prospective, randomized trial at a 650-bed tertiary medical center hospital.Fifty-one consecutive CMV-seronegative patients who received renal allografts from seropositive donors between March 1990 and April 1992.Patient and allograft survival, and the incidence and severity of CMV disease.Cytomegalovirus prophylaxis with seven doses of intravenous immunoglobulin for 6-week periods (group 1, n = 27) or low-dose intravenous ganciclovir for 3 weeks (group 2, n = 24). Results were compared with those obtained in 23 CMV-seronegative historical controls who received renal allografts from CMV-seropositive donors between 1987 and 1989, and who did not receive prophylaxis for CMV (group 3).Both prophylactic regimens significantly reduced the incidence of invasive CMV infection (P.05) and were well tolerated. However, the cost of ganciclovir ($350 per patient) was substantially less than that of immunoglobulin ($4000 per patient).These data suggest that prophylactic ganciclovir therapy provides a cost-effective approach toward significantly improving the outcome of renal transplantation in recipients at risk for primary CMV disease.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), concanavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA greater than M17 greater than M1 greater than M21 much greater than M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and M1 inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.

The thiol-alkylating agent, N-ethylmaleimide (NEM), was found to inhibit the response of human peripheral blood mononuclear cells to the T-cell mitogen, concanavalin A (Con A). NEM (10 microM) blocked Con A-induced agglutination, production of interleukin-2 (IL-2) and expression of the IL-2 receptors (Tac) without toxicity. In order to determine whether the effects of NEM on lymphokine production were related to inhibition of agglutination, we compared the immunosuppressive effects of NEM with those of cytochalasin A, cytochalasin B and colchicine. NEM did not inhibit E-rosette (ER) formation, suggesting that it does not interfere with actin filaments. Low concentrations of NEM (4 microM) inhibited IL-2 production and Tac expression without inhibiting agglutination, while 6-10 microM NEM blocked agglutination and DNA synthesis as well. In contrast, 5-10 microM cytochalasin B (CB) inhibited ER formation, agglutination, Tac expression and DNA synthesis, but augmented IL-2 production by three- to ten-fold. Colchicine (0.1-10 microM) had no effect on ER formation or agglutination and augmented IL-2 production by as much as 18-fold. However, colchicine blocked Tac expression by greater than 40% and DNA synthesis by greater than 80%. Cytochalasin A (CA), which has the thiol-reactive properties of NEM, the actin filament-disrupting properties of CB, and the microtubule-disrupting properties of colchicine, exhibited the immunosuppressive effects of all three compounds. These studies suggest that the inhibitory effects of NEM on IL-2 production do not appear to be due to reactivity with the cytoskeleton, but are probably due to effects on signal transduction pathways leading to IL-2 production and expression of IL-2 receptors.

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [3H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [3H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10(7) BW5147 transplanted leukemia cells had 130 +/- 12 units of AKR-LSF activity/ml of serum compared to 40 +/- 8 units/ml for mice with spontaneous leukemia. Normal mouse serum contained 33 +/- 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.

• Hydrogen peroxide, a reactive oxygen intermediate produced by activated neutrophils, has been shown to inhibit the response of human T lymphocytes to mitogens and alloantigens. Since hydrogen peroxide is known to react with iron and to induce lipid peroxidation, we compared the effects of hydrogen peroxide and a lipid peroxidation product, malondialdehyde, on the response of human peripheral blood mononuclear cells to T-cell mitogens. Peripheral blood mononuclear cells pretreated with 1 mmol/L of malondialdehyde, washed, and resuspended in fresh medium exhibited no inhibition of phytohemagglutinin responsiveness. Peripheral blood mononuclear cells treated in the same manner but with 200 μmol/L of hydrogen peroxide were inhibited by more than 95%. The addition of ferric edetate did not alter the inhibitory effects of 50 to 100 μmol/L of hydrogen peroxide, nor did the addition of deferoxamine, an iron chelator. These studies suggest that exogenous lipid peroxidation does not affect lymphocyte activation but that hydrogen peroxide has a direct inhibitory effect. Although monocytes are necessary for T-cell mitogenic responses, the effect of hydrogen peroxide was found to be directed at T lymphocytes. Exposure of T cells to a single dose of 200 μmol/L of hydrogen peroxide resulted in more than 71% suppression of the proliferative response measured 48 hours later, but the effect was spontaneously reversed by 72 to 96 hours. Repeated exposure of the cells to hydrogen peroxide resulted in continued inhibition of the proliferative response. These findings suggest that hydrogen peroxide produced by inflammatory phagocytic cells might be capable of suppressing the immune response of nearby T lymphocytes. ( Arch Surg 1988;123:300-304)

• Hydrogen peroxide (100 to 200μM) inhibited the response of human peripheral-blood mononuclear cells (PBMCs) in phytohemagglutinin, concanavalin A, and mixed lymphocyte culture assays by more than 90% without affecting cell viability. The response of PBMCs to pokeweed mitogen was stimulated twofold by 50μM H 2 O 2 , but 200μM H 2 O 2 inhibited the pokeweed mitogen response by more than 95%. A 50μM concentration of H 2 O 2 completely blocked the generation of cytotoxic T cells in the mixed lymphocyte culture but did not inhibit the production of interleukin 2. Concentrations of 100μM to 200μM H 2 O 2 inhibited interleukin 2 production by 45% to 57%. The H 2 O 2 appeared to block early events in T-cell activation, since 200μM H 2 O 2 was not inhibitory when added one hour after stimulating the cells with phytohemagglutinin. Treatment of PBMCs with 200μM H 2 O 2 did not decrease the total cellular thiol pool, suggesting that H 2 O 2 -mediated inhibition of the proliferative response was not due to thiol oxidation. However, pretreatment of PBMCs with the lipid antioxidants butylated hydroxyanisole, butylated hydroxytoluene, and n -propyl gallate blocked more than 75% of the inhibitory effect of H 2 O 2 , suggesting that H 2 O 2 inhibits T-cell activation by inducing lipid peroxidation. ( Arch Surg 1987;122:99-104)