Initial experience of dual maintenance immunosuppression with steroid withdrawal in vascular composite tissue allotransplantation.

Current immunosuppression in VCA is largely based on the experience in solid organ transplantation. It remains unclear if steroids can be reduced safely in VCA recipients. We report on five VCA recipients who were weaned off maintenance steroids after a median of 2 months (mean: 4.8 months, range 2-12 months). Patients were kept subsequently on a low dose, dual maintenance consisting of tacrolimus and mycophenolate mofetil/mycophenloic acid with a mean follow-up of 43.6 months (median = 40 months, range 34-64 months). Early and late acute rejections responded well to temporarily augmented maintenance, topical immunosuppression, and/or steroid bolus treatment. One late steroid-resistant acute rejection required treatment with thymoglobulin. All patients have been gradually weaned off steroids subsequent to the treatment of acute rejections. Low levels of tacrolimus (<5 ng/mL) appeared as a risk for acute rejections. Although further experience and a cautious approach are warranted, dual-steroid free maintenance immunosuppression appears feasible in a series of five VCA recipients.

TIRC7 and HLA-DR axis contributes to inflammation in multiple sclerosis.

BACKGROUND AND OBJECTIVE: Interactions between TIRC7 (a novel seven-transmembrane receptor on activated lymphocytes) and its ligand HLA-DR might be involved in the inflammatory process in multiple sclerosis (MS). METHODS: Methods comprised immunohistochemistry and microscopy on archival MS autopsies, proliferation-, cytokine-, and surface-staining assays using peripheral blood lymphocytes (PBLs) from MS patients and an in vitro model. RESULTS: TIRC7 was expressed in brain-infiltrating lymphocytes and strongly correlated with disease activity in MS. TIRC7 expression was reduced in T cells and induced in B cells in PBLs obtained from MS patients. After ex vivo activation, T cell expression of TIRC7 was restored in patients with active MS disease. The interaction of TIRC7(+) T lymphocytes with cells expressing HLA-DR on their surface led to T cell proliferation and activation whereas an anti-TIRC7 mAb preventing interactions with its ligand inhibited proliferation and Th1 and Th17 cytokine expression in T cells obtained from MS patients and in myelin basic protein-specific T cell clone. CONCLUSION: Our findings suggest that TIRC7 is involved in inflammation in MS and anti-TIRC7 mAb can prevent immune activation via selective inhibition of Th1- and Th17-associated cytokine expression. This targeting approach may become a novel treatment option for MS.

Two genes required for diabetes in BB rats. Evidence from cyclical intercrosses and backcrosses.

The BB rat develops a syndrome of autoimmune diabetes similar to Type I diabetes of man. It also has a severe T cell lymphopenia. As part of an ongoing breeding program to transfer the diabetogenic genes of the BB rat onto inbred rat strain backgrounds, diabetic animals were used in a backcross (BC)- intercross (IC)-backcross breeding scheme with Brown Norway (BN), Lewis (L), and Wistar-Furth (WF) inbred rats. We have used monoclonal antibodies to analyze both lymphopenia and major histocompatibility (MHC) antigens (the RT1 locus in the rat) in relation to the development of diabetes. To examine T cell subsets we used a panel of monoclonal antibodies, in particular W3/25 and OX19 , which discriminate the abnormal phenotype better than W3/13. In our breeding program, at least two independent genes or gene complexes are required for the expression of diabetes. One gene determines the lymphopenia, is inherited by simple autosomal recessive genetics and is not linked to the MHC. The second gene is linked to the MHC. Both genes are necessary, but neither gene is sufficient by itself for the development of diabetes.

Detecting antibodies with similar reactivity patterns in the HLDA8 blind panel of flow cytometry data.

The blind panel collected for the 8th Human Leucocyte Differentiation Antigens Workshop (HLDA8; ) included 49 antibodies of known CD specificities and 76 antibodies of unknown specificity. We have identified groups of antibodies showing similar patterns of reactivity that need to be investigated by biochemical methods to evaluate whether the antibodies within these groups are reacting with the same molecule. Our approach to data analysis was based on the work of Salganik et al. (in press) [Salganik, M.P., Milford E.L., Hardie D.L., Shaw, S., Wand, M.P., in press. Classifying antibodies using flow cytometry data: class prediction and class discovery. Biometrical Journal].

A compendium of gene expression in normal human tissues.

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Selective sparing of suppressor cells generated in mixed lymphocyte response by an anti-interleukin-2 receptor antibody.

Recent investigations have shown that anti-IL-2 receptor antibodies can prolong cardiac allograft survival in animal models and can be used effectively as primary immunosuppressive therapy in human renal transplantation. While previous studies have established that helper and cytotoxic T cells require IL-2 for proliferation, the role of this lymphokine in suppressor cell development is uncertain. We therefore studied the effects of SA36.6G (a monoclonal antibody directed at the 55 kD chain of the high-affinity IL-2 receptor) on events occurring in the mixed lymphocyte reaction. As expected, when added at culture initiation, SA36.6G inhibited both the proliferative response to allogeneic stimulation, and the generation of cytotoxic T cells. These effects were not the result of altered growth kinetics. In contrast, the generation of suppressor cells with a polymorphic pattern of specificity was not blocked by SA36.6G. Cultures containing SA36.6G had decreased numbers of activated lymphoblasts, but among this activated cell population the proportion of 2H4+ cells was doubled (53 +/- 13% vs. 27 +/- 9%). SA36.6G also blocked the appearance of IL-2 receptors on activated cells as determined by flow cytometry. This relative sparing of suppressor cells by an anti-IL-2 receptor antibody suggests that these cells may either exhibit IL-2 independent proliferation, or utilize an IL-2 receptor not recognized by SA36.6G.

Requirements for the induction of allospecific CD8+ suppressor T cells in the rat primary mixed lymphocyte response. CD4+, CD45R+ T cells, or supernatant factor.

We examined the requirements for the induction of the MLR-generated allospecific CD8+ suppressor T cells in the rat. Depleting the responder population of CD4+ T cells before initiating the primary MLR abrogates the generation of day-5 CD8+ T suppressor effectors. Readdition of at least 10% CD4+ T cells to the CD4+ depleted primary MLR reconstitutes suppressor cell generation. Using the anti-CD45R monoclonal antibody OX22, we also show that the T suppressor inducer cells are CD4+ CD45R+. Using a dual chamber Transwell culture system, which allows cells to be co-incubated without direct cell-to-cell contact, we show that a soluble factor/s, produced during the course of the primary MLR, is capable of inducing naive CD8+ T cells to become suppressor effectors but only when these CD8 T cells are in direct contact with allogeneic stimulators. Allospecificity is conferred by the stimulator cells and not by the suppressor-inducer factor. The supernatant of day-5 primary MLR is also capable of inducing antigen-specific suppressor effectors from naive CD8+ T cells, and also only in the presence of allogeneic stimulator cells. Recombinant human IL-2, in doses that are up to five times the amount present in the supernatant cultures, is unable to induce suppressor-effector cells from naive CD8+ T cells. We conclude that, to become allospecific suppressor effectors, naive CD8+ T cells require contact with allogeneic stimulator cells and either CD4+ CD45R+ suppressor inducer cells or suppressor inducer factor/s produced during the course of the primary MLR.

Interleukin 2 receptor expression on peripheral blood lymphocytes in association with renal allograft rejection.

Periodic assay of IL-2 receptor expression on the surfaces of peripheral blood lymphocytes might provide information predictive of in vivo immunologic events. This study compares two methods of determining IL-2 receptor expression after renal transplantation in cynomolgus monkeys. The first utilized single color staining of peripheral blood mononuclear cells with mouse anti-human IL-2 receptor monoclonal antibody followed by a fluorescein-labeled goat anti-mouse IgG antibody. Epics C cell sorter windows were set to count cells of the size and granularity of normal lymphocytes. The second utilized two-color staining with fluorescein-labeled anti-IL-2 receptor antibody, combined with phycoerythrin-labeled anti-CD4 antibody or with phycoerythrin-labeled anti-CD8 antibody. Two-color staining allowed the sorter windows to be enlarged to count all mononuclear cells, regardless of size or granularity, without introducing the contaminating effects of monocytes. Data obtained from single-color staining showed no consistent or significant expression of the IL-2 receptor on peripheral lymphocytes in association with the rejection process. Data obtained from two-color staining revealed an increase of IL-2 receptor expression on peripheral T cells of at least 10% from the postoperative baseline, which preceded the creatinine rise from allograft rejection in 13 of 13 animals. Increases in IL-2 receptor expression on T cells were not specific to rejection, however. Some animals in which treatment produced a delay of rejection showed a transient rise in IL-2 receptor expression around post-transplant day 5, which was not followed by a rise in creatinine. The two-color staining technique described provides a sensitive means of detecting IL-2 receptor expression in vivo and documents the association of increases in IL-2 receptor expression on T cells with rejection.

HLA gene and haplotype frequencies in the North American population: the National Marrow Donor Program Donor Registry.

BACKGROUND: As of May 1, 1995, the National Marrow Donor Program had a donor registry consisting of over 1.35 million HLA-typed volunteers recruited from most major cities and states in the United States. This registry represents the largest single HLA-typed pool of normal individuals in the world. METHODS: We analyzed the HLA-A, -B, -DR locus phenotypes of the National Marrow Donor Program donors in order to estimate gene and haplotype frequencies for major racial groups of the United States: Caucasian American, Asian American, African American, Latin American, and Native American. The large size of the database allowed us to calculate the frequencies of relatively rare antigens and haplotypes with more accuracy than previous studies. RESULTS: We observed 89,522 distinguishable HLA-A, -B phenotypes in 1,351,260 HLA-A, -B-typed donors and 302,867 distinguishable HLA-A, -B, -DR phenotypes in 406,503 HLA-A, -B, -DR-typed donors. Gene and haplotype frequencies differed remarkably among the five racial groups, with African Americans and Asian Americans having a large number of haplotypes that were specific to their racial groups, whereas Caucasian Americans, Latin Americans, and Native Americans shared a number of common haplotypes. CONCLUSIONS: These data represent an important resource for investigators in the fields of transplantation and population genetics. The gene and haplotype frequencies can be used to aid clinicians in advising patients about the probability of finding a match within a specific ethnic group, or to determine donor recruitment goals and strategies. The information is also a valuable resource for individuals who are interested in population genetics, selection and evolution of polymorphic human genes, and HLA-disease association.

Expression of RT1.A and RT1.B/D antigens on endothelium of rat kidneys.

The presence of major histocompatibility complex (MHC)-encoded antigens (RT1) on endothelium of rat kidneys was investigated using hyperimmune allogeneic rat sera (HIS) in an indirect immunofluorescence technique with kidney sections. Endothelial antigens detected with anti-MHC sera segregated in an F2 generation with the RT1 antigens on erythrocytes. The MHC specificity of the endothelial staining was established with kidneys from congenic strains. The existence of public and private determinants on endothelium was shown by absorption experiments with spleen cells from several strains. RT1.A antigens were demonstrated on the endothelium of arteries, capillaries, and veins, using purified anti-RT1.A antibodies. This was confirmed using the MHC-recombinant PVG.1R strain. RT1.B/D antigens were found on the endothelium of capillaries and veins of the same strain. Furthermore, red blood cell-absorbed HIS, monoclonal anti-Ia antibodies, and mouse alloantisera which cross-react with rat Ia-like antigens were used. Based on this cross-reactivity, it appeared that I-E/C-like but not I-A-like antigens were detected on endothelium. RT1.C region antigens were not detected. Antisera against RT2, Ag-F, Ag-S, and immunoglobulin allotypes did not bind to normal kidneys. We conclude that RT1.A and some RT1.B/D specificities are expressed on rat renal endothelium.

Angiotensin gene polymorphism as a determinant of posttransplantation renal dysfunction and hypertension.

BACKGROUND: Polymorphism of the genes associated with angiotensin, including angiotensin-converting enzyme (ACE), angiotensinogen (AGT), and the type 1 (AT1) and type 2 (AT2) angiotensin II receptors, has been implicated in the pathophysiology of hypertension, ischemic heart disease, and progression of chronic renal disease. METHODS: We investigated the impact of the ACE, AGT, AT1, and AT2 genotypes on renal allograft function in 148 patients (77 men, 71 women) who underwent transplantation over a 5-year period. Patients were genotyped using polymerase chain reaction sequence-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. RESULTS: ACE (D) and AGT (A/A) genotypes were associated with poorer chronic renal transplant function and more rapid chronic progression, defined as an increase of serum creatinine level with time. In addition, mean diastolic blood pressure at 3 years was significantly (P<0.02) correlated with C gene dose of AT1 (A-->C, 1166), with levels of 79+/-10 mmHg, 82+/-8.6 mmHg, and 95+/-8.3 mmHg for the A/A, A/C, and C/C genotypes, respectively. An apparent AT2 homozygote disadvantage could be an epiphenomenon because AT2 maps to the X chromosome, and males are homozygous for just one of the AT2 alleles (A/- or G/-). CONCLUSIONS: Pretransplantation testing of the ACE, AGT, and AT1 genotypes may assist clinicians in identifying patients at risk for chronic renal transplant dysfunction and hypertension.

Presensitization and the renal allograft recipient.

We examined data from the New England Organ Bank to characterize the influence of patient sensitization on allograft survival, and our current crossmatching strategy. To evaluate our recipient eligibility criteria, we compared computer-predicted crossmatch results to 3622 actual crossmatches. A computer-predicted positive crossmatch was highly predictive of an actual positive crossmatch, for patients with a percentage reactive antibody of 40% of more (positive predictive value 91-99%), thus obviating the need to perform the actual crossmatch. Given the high prevalence of sensitized patients on our waiting list, very few individuals are inappropriately excluded from consideration for an available organ. In contrast, a negative computer prediction was never sufficiently predictive of a negative crossmatch to dispense with the actual crossmatching procedure. We also compared graft survival in patients with positive antidonor crossmatches using historical (greater than 6 months old) sera with those with negative historical crossmatches (or with no history of humoral sensitization). One-year actuarial graft survival in the first group was 61.0 +/- 6.0%, compared with 85.2 +/- 1.4% in those without positive historical crossmatches (P less than 0.001). This adverse effect of a positive historical crossmatch was true in both first transplants (n = 41, 1-year graft survival 67.9 +/- 7.4% vs. 86.2 +/- 1.6%, P less than 0.05) and in regrafted individuals (n = 29, 1-year graft survival 50.7 +/- 9.8% vs. 78.9 +/- 3.7%, P less than 0.01). The inability to accurately predict negative crossmatches, and the possible adverse effect of positive historical crossmatches on graft survival, represent potential obstacles to a goal of national organ sharing.

Differential role of CD4+ cells in the sensitization and effector phases of accelerated graft rejection.

Although CD4-targeted therapy markedly prolongs survival of organ allografts in naive rodents, its effects in primed hosts have not been studied. In our model of accelerated rejection (ACCR) of cardiac Tx in rats, treatment with BWH-4, a CD4 mAb (IgG2a), in the sensitization (between skin and heart Tx) but not in the effector (after cardiac Tx) phase, abrogated fulminant less than 36 hr rejection response and prolonged Tx survival to ca. 11 days. This effect correlated with decreased frequency of circulating CD4+ cells, but it did not depend upon their total depletion. It was also related to BWH-4 mAb-mediated elimination/depression of strong anti-donor humoral responses and cellular responses as determined by lymphocyte-mediated cytotoxicity and mixed lymphocyte reaction and mounted otherwise at the time of engraftment by untreated sensitized hosts. Immunoperoxidase studies of cardiac Tx from BWH-4-conditioned recipients revealed reduced T and B cell activities, reflected in abolition/reduction in deposition of humoral mediators, infiltrating cells, intra-Tx elaboration of interleukin-2 and interferon-gamma, and cell activation. This first report of the successful use of CD4 mAb in sensitized recipients of vascularized organ Tx, stresses the role of CD4+ cells as potential targets for immunosuppression in the sensitization phase of accelerated Tx injury. The beneficial therapeutic effect, probably due to both depletion and functional inhibition of CD4+ T cells, has been achieved by using relatively low doses of BWH-4 mAb.

Evidence that therapeutic strategies targeted at CD4+ cells modulate accelerated rejection of cardiac allografts in sensitized rats by different mechanisms.

(LEW x BN)F1 cardiac allografts are rejected within 36 hr in LEW rats presensitized with BN skin grafts 7 days earlier (acute rejection occurs within 8 days). We have previously described the effects of individual CD4 (BWH-4), CD25 (IL-2R, ART-18) mAbs, and CsA therapeutic regimens upon cardiac allograft survival in sensitized hosts. The present studies were designed to probe an adjunctive use of ART-18 or CsA upon BWH-4-mediated suppression of accelerated graft injury. Sequential therapy with BWH-4 and ART-18 in the sensitization phase (days -7 to -1) and effector phase (from day 0, the day of cardiac transplant), respectively, prolonged graft survival additively to c. 22 days. Treatment with BWH-4 markedly diminished host humoral response against ART-18 preparation. BWH-4 given in concert with subtherapeutic dose of CsA produced graft survival comparable to that induced by mAb alone (c. 13 days) with concomitant decreased host anti-BWH-4 response. None of the combined regimens affected the frequency of circulating CD4+ cells, as compared with that exerted by BWH-4 monotherapy. Thus this study defines principles and some mechanistic aspects of optimal immunosuppressive strategies potentiating the effects of CD4-targeted therapy.

Decrease in phenotypically defined T helper inducer cells (T4+4B4+) and increase in T suppressor effector cells (T8+2H4+) in stable renal allograft recipients.

Two monoclonal antibodies, anti-2H4 and anti-4B4, reciprocally divide the T4+ (CD4+) and T8+ (CD8+) lymphocytes into T4+2H4+, T4+4B4+, T8+2H4+ and T8+4B4+ subsets. The T4+2H4+, T4+4B4+ and T8+2H4+ subsets possess suppressor-inducer, helper-inducer, and suppressor-effector function, respectively, as previously defined in a system of B cell immunoglobulin production. Using monoclonal antibodies, including anti-2H4 and anti-4B4, and flow cytometry, we monitored lymphocyte subpopulations in 66 renal allograft recipients. We found that patients with stable allograft function have a decrease in the percentage of total T4+ lymphocytes from 41.9 +/- 9.5% pretransplant (pre-Tx) to 36.3 +/- 13.9% posttransplant (post-Tx) (P less than 0.05). This decrease was seen mainly in the T4+4B4+ or helper-inducer subset from 20.8 +/- 4.7% (pre-Tx) to 16.0 +/- 6.3% (post-Tx) (P less than 0.005). Patients with stable function were also noted to have an increase in the percentage of total T8+ lymphocytes from 21.3 +/- 10.7% (pre-Tx) to 30.9 +/- 15.4% (post-Tx) (P less than 0.02). Examination of T8 subsets revealed that a statistically significant increase was seen in the T8+2H4+ or suppressor effector subset from 15.5 +/- 9.2% (pre-Tx) to 21.5 +/- 10.2% (post-Tx) (P less than 0.01). Additionally, serial studies on 14 patients revealed an increase in the %T4+2H4+ suppressor-inducer subset from 9.31 +/- 3.64% (pre-Tx) to 15.71 +/- 6.41% (post-Tx) (P less than 0.0025). Since the role of these subsets has not been established in alloimmunity, in vitro allogeneic studies of 2H4-enriched (2H4+) and 2H4-depleted (2H4-) lymphocytes from normal peripheral blood were performed. In the mixed lymphocyte reaction, 2H4+ cells proliferated less than 2H4- cells (cpm ratio 2H4+/2H4-: 0.63-0.84), but 2H4+ cells generated twice as much suppressor activity as 2H4- cells (ratio % suppression 2H4+/2H4-: 1.9-2.3). These results suggest that 2H4+ cells play a role in the suppressor limb of the alloimmune response and that the increase in cells of this phenotype in our transplant population may be responsible for the maintenance of stable allograft function.

Blocking of mononuclear cell accumulation, cytokine production, and endothelial activation within rat cardiac allografts by CD4 monoclonal antibody therapy.

CD4 monoclonal antibody therapy prolongs allograft survival in a variety of experimental models and is currently undergoing clinical trials, though surprisingly little is known about the effects of CD4 mAb therapy on intragraft effector mechanisms that mediate rejection. We previously reported the significantly improved survival of (LEWxBN)F1 cardiac allografts in LEW rats treated for 10 days with the new CD4 mAb, BWH-4, at a dose of 700 micrograms/day, i.v., starting at the time of engraftment. Thus, CD4-treated rats showed prolongation of allograft survival to a median of 37 days (range 22 to greater than 100 days) post-Tx, compared with rejection at 7 days in untreated controls. We now report the results of detailed immunohistologic studies of allografts collected from these rats. Comparison of acutely rejecting allografts in untreated rats with well-functioning allografts collected at day 7 post-Tx from CD4-treated rats showed that CD4 mAb: (1) significantly reduced mononuclear cell infiltration, interstitial edema, hemorrhage formation and vascular and extravascular thrombosis; (2) inhibited mononuclear cell induction of receptors for IL-2 and transferrin, and upregulation of class II antigens and ICAM-1 on leukocytes and endothelial cells; (3) suppressed intragraft mononuclear cell and/or endothelial production of the cytokines IL-1, IL-2, IL-6, IFN-gamma, and TNF; and (4) blocked upregulation of endothelial tissue factor and downregulation of thrombomodulin, and consequently inhibited fibrin deposition. Studies of allografts from CD4-treated rats collected at day 30 post-Tx, prior to clinical rejection, showed a resurgence of CD4+ cells within allografts and a dense cellular immune response. We conclude that short-term CD4 mAb therapy has potent and extensive inhibitory effects on cytokine-related mononuclear cell and endothelial activation in vivo, blocking multiple afferent and efferent steps of the alloresponse.

Conversion from cyclosporine to azathioprine in renal allograft recipients.

Fifty seven recipients of renal allografts initially treated with CsA and low-dose prednisone were switched to azathioprine and low-dose steroids. Ten had prolonged (greater than 28 days) allograft nonfunction after transplantation (group 1), 8 had ongoing, poorly controlled rejection (group 2), and 39 had stable functioning grafts (group 3). With a mean follow-up period of 5 +/- 3 months after conversion, 50 grafts remained functional including 6 of 10 in group 1, 6 of 8 in group 2, and 38 of 39 in group 3. Thirty-seven (65%) had improved function, 12 (21%) had stable function, and 8 (14%) experienced declining renal function. Three of these latter 8 patients required reinstitution of CsA therapy. There were 20 episodes of acute rejection in 18 patients; one graft lost function because of acute rejection unresponsive to therapy. Reasons for the 6 other graft losses were persistent primary nonfunction in 3 patients from group 1, untreated rejection in 2 patients who had multiple prior rejection episodes while on CsA, and chronic rejection in one patient. Although renal function has improved or stabilized in the majority (86%) of individuals changed to azathioprine therapy, there was substantial risk of acute rejection (32%) complicating this procedure. Patients most likely to benefit from conversion to azathioprine therapy are those with prolonged graft nonfunction after transplantation and those with serum creatinines greater than 2.0 mg/dl.

Effects of BWH-4 anti-CD4 monoclonal antibody on rat vascularized cardiac allografts before and after engraftment.

We studied the effects and mechanism of action of BWH-4, an IgG2a mouse antirat CD4 monoclonal antibody that recognizes a distinct epitope on the CD4 molecule, in LEW recipients of (LEW x BN)F1 vascularized heterotopic cardiac allografts. Ten animals received daily injections of 700 micrograms BWH-4 for ten days after engraftment. Median actuarial allograft survival was 37 days for the treated animals compared with 7.5 days for controls (n = 6). There was depletion of peripheral blood CD4+ lymphocytes to 4 +/- 1% one week posttransplant (normal = 48 +/- 4%, n = 6). Six additional animals were treated with a lower dose (100 micrograms) of BWH-4 daily for ten days. Median actuarial allograft survival was 10 days and the circulating CD4+ cells decreased to 30 +/- 6% at one week posttransplant. All six low-dose-treated animals mounted an anti-BN alloantibody response by 3 weeks, while only one of the ten high-dose-treated animals had positive alloantibodies two weeks posttransplant. Lymphocyte-mediated cytotoxicity against donor lymphoblasts was completely abolished in the high-dose-treated animals when compared with acutely rejecting controls. We also used low-dose (100 micrograms) BWH-4 to pretreat eleven experimental animals for 7 days prior to engraftment. The circulating CD4+ cells decreased to only 12 +/- 2% on the day of transplantation and 26 +/- 9% one week posttransplant. However, six (55%) pretreated animals survived more than 55 days. There was a 66% decrease in lymphocyte-mediated cytotoxicity against donor lymphoblasts when compared with acutely rejecting controls. We conclude that the anti-CD4 mAb, BWH-4, prevents acute rejection of vascularized heterotopic rat cardiac allografts; this effect is mediated by depletion of the CD4+ T cell subset, suppression of alloantibody production, and inhibition of lymphocyte-mediated cytotoxicity.

Prevention of graft-versus-host disease following small bowel transplantation with polyclonal and monoclonal antilymphocyte serum. The effect of timing and route of administration.

Graft-versus-host disease is a potential problem following small bowel transplantation. We have previously shown that a two-day intraperitoneal course of polyclonal antilymphocyte serum completely prevents GVHD without impairing allograft function in a unidirectional rat small bowel transplant model. In the present study we sought to determine the optimum route and timing of ALS administration and whether donor pretreatment with the anti-T cell receptor monoclonal antibody R73 would be similarly effective in preventing GVHD. Both intravenous and intraperitoneal injection of ALS effectively prevent GVHD in this model. ALS must be given to donors at least 48 hr prior to graft procurement for maximum effectiveness. Prevention of GVHD correlates with lymphocyte depletion in mesenteric lymph nodes, as opposed to peripheral blood or small bowel lamina propria. Donor pretreatment with the monoclonal antibody R73 significantly delays the onset of GVHD in this small bowel transplant model but appears less effective than polyclonal ALS.

Evidence that monoclonal antibodies against the 55kD subunit of the rat IL-2 receptor do not inhibit the development of suppressor cells generated in mixed lymphocyte culture.

Although the ability of Ts to prevent allograft rejection has been well established, their intrinsic characteristics and dependence upon lymphokines remain poorly defined. The cells from unmodified LEWxBN bulk 5-day rat MLR inhibit both proliferation in test MLR and generation of CTL, as well as prolonging the survival of donor-specific test cardiac allografts following adoptive transfer. We have examined the effects of a panel of mAb directed against functionally distinct epitopes on the p55 subunit of rat IL-2R on the generation and in vitro/in vivo activity of MLR-generated Ts. ART-18 (which blocks IL-2-dependent T cell growth) was the only mAb from the panel that profoundly suppressed alloreactive T cell proliferation in primary MLR (47.5%). However, the generation of Ts was never affected by any mAb (% suppression in test MLR = 40-60%). Neither ART-18 nor ART-65 (which does not affect T cell proliferation) interfered with the efficacy of Ts to inhibit CTL generation in fresh bulk MLR. Adoptive transfer of cells (3-10 x 10(6] from ART-18 or ART-65-modulated MLR into naive LEW rats prolonged (LEW x BN)F1 test cardiac allograft survival to 11-13 days (P less than 0.05 as compared with acutely rejecting hosts). All in vitro and in vivo effects exerted by MLR-generated cells were antigen-specific. In unmodified MLR, Ts were IL-2R+ (ca. 50% of total blasts), as shown by cell separation using magnetic beads. In contrast, in MLR with ART-18 added, Ts were primarily IL-2R- (ca. 10% of blasts). Thus, antirat p55 subunit IL-2R mAb do not inhibit MLR-generated Ts functionally operative in vitro and in vivo. IL-2R- Ts precursors requiring lymphokine(s) other than IL-2 may differentiate into IL-2-dependent Ts effectors. Such divergent IL-2 requirements for Ts growth in vitro may explain the Ts-sparing effects in allograft recipients treated with anti-IL-2R mAb.