Impaired autologous and allogeneic mixed lymphocyte reaction in human marrow transplant recipients.

The autologous and allogeneic mixed lymphocyte reactions of enriched T to non-T cells from 54 allogeneic marrow recipients were studied. Reactivity of cells from almost all patients was severely impaired when compared with those from their marrow donors. There were no differences between lymphocyte responses of patients with and without graft-versus-host disease. However, in the autologous mixed lymphocyte reaction the impairment was more severe in cells from short-term than in those from long-term patients. Coculture experiments using enriched T or non-T cells from patients mixed with T or non-T cells from their respective marrow donors indicated that the impairment resided primarily in the responding T cells, partially in stimulating non-T cells, and perhaps also in the interactions of T and non-T cells. In the allogeneic mixed lymphocyte reaction, T cells from patients showed defective responses to normal allogeneic non-T cells. Their non-T cells, however, often had normal allogeneic stimulating capacity.

Two monoclonal antibodies (DLy-1 and DLy-6) directed against canine lymphocytes.

Two murine monoclonal antibodies directed against canine lymphocytes are described. DLy-1, raised against puppy thymocytes, and DLy-6, raised against bronchoalveolar lymphocytes, both react with most lymphocytes in peripheral blood, thoracic duct lymph, and bronchoalveolar lavage fluids. DLy-1 also recognizes monocytes and granulocytes. However, it is not reactive with erythrocytes, megakaryocytes, or platelets. Expression of DLy-1 antigen on thymocytes ranged from 5--30%. The distribution of DLy-6 antigens seems to be confined to lymphoid cells. Ten to 60% puppy thymocytes were positive. Interestingly, lymphoblasts formed in response to stimulation with mitogens or alloantigens lacked DLy-6 in contrast to DLy-1 cell surface antigen expression.

Autologous marrow recovery and sensitization to non-HLA antigens after HLA-identical marrow transplantation for aplastic anemia.

One hundred seventy-five patients with severe aplastic anemia were treated by high-dose cyclophosphamide and HLA-A, -B, and -D-identical sibling marrow transplants. Thirty-eight patients rejected their grafts. Four of the 38 showed autologous marrow recovery as determined by blood genetic markers. The remarkable feature of one case following autologous marrow recovery was the presence of unidirectional proliferative and cytotoxic responses of circulating host lymphocytes to marrow donor lymphocytes in mixed lymphocyte culture and cell-mediated lympholysis. Presumably these responses were the result of in vivo sensitization to those non-HLA antigens for which donor and recipient differed.

Enrichment (and depletion) of human suppressor cells with monoclonal antibodies and immunoglobulin-coated plates.

A cell separation method using immunoglobulin (Ig)-coated plates, originally devised for murine spleen cells, was modified and adapted for enrichment (and depletion) of cellular subpopulations from human peripheral blood. For the direct separation of B and T cells, F(ab')2 fragments of anti-human Ig were used to coat the plates. For indirect separation, the cells were first incubated with monoclonal antibodies to cell surface antigens and then separated in plates coated with anti-mouse Ig. Plates were first coated with poly-L-lysine to facilitate the adherence of anti-Ig antibodies, and finally with bovine serum albumin to mask free poly-L-lysine. Cells which did not react with the anti-Ig antibodies or which were nonadherent to the plate were pipetted off; cells which reacted with the anti-Ig antibodies or which were adherent were eluted after incubation with excess serum. T, non-T, T4+, T4-, T8+, and T8- lymphocytes were separated with high viability, purity, and yield. The method was used to study suppressor activity of a patient who was treated by bone marrow transplantation for myelofibrosis. Strong suppressor activity was associated with unfractionated peripheral blood mononuclear leukocytes, monocytes, T, T8+, and T4- cells but not with B, T8-, and T4+ cells.

Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients.

This study of chronic graft-versus-host disease (GVHD) describes the clinical, pathologic and laboratory features, and the causes of morbidity and mortality in 20 patients who received allogeneic marrow transplants from HLA identical sibling donors. Chronic GVHD is a pleiotrophic syndrome with variability in the time of onset, organ systems involved and rate of progression. The clinical-pathologic features resemble an overlap of several collagen vascular diseases with frequent involvement of the skin, liver, eyes, mouth, upper respiratory tract, esophagus and less frequent involvement of the serosal surfaces, lower gastrointestinal tract and skeletal muscles. Major causes of morbidity are scleroderma with contractures and ulceration, dry eyes and mouth, pulmonary insufficiency and wasting. Chronic GVHD has features of immune dysregulation with elevated levels of eosinophils, circulating autoantibodies, hypergammaglobulinemia and plasmacytosis of viscera and lymph nodes. In this study, three patients had limited chronic GVHD with relatively favorable prognosis characterized by localized skin involvement and/or hepatic disease without chronic aggressive histology. Most patients, however, had extensive disease with a progressive course. Survival was largely determined by the presence or absence of serious recurrent bacterial infections. The over-all severity of disease was best assessed by using the Karnofsky performance rating.

Fibronectin restores defective in vitro proliferation of patients' lymphocytes after marrow grafting.

In search for means of improving the impaired lymphocyte function of recipients after marrow grafting, we investigated the effect of fibronectin (FN) on patients' lymphocytes in allogeneic mixed lymphocyte cultures (MLC) and in cell-mediated lympholysis (CML) assays, since these tests are usually defective in transplanted patients. Four subgroups of marrow recipients were tested: patients within the first 100 days of transplantation (short-term) with (n = 16) or without (n = 14) acute graft-versus-host disease (GVHD), and long-term recipients with (n = 23) or without (n = 15) chronic GVHD. Exogenous FN (25 micrograms/ml) increased the proliferative response in the allogeneic mixed lymphocyte culture (MLC) significantly in cells from short-term patients without acute GVHD (+42%) and in those from long-term recipients with (+117%) and without chronic GVHD (+48%). In cells from patients with chronic GVHD, 3H-thymidine uptake after the addition of FN was enhanced to the level of that in lymphocytes of the corresponding marrow donor without exogenous FN. Fibronectin was effective only if added at the beginning of the MLC. In contrast to the results in MLC, exogenous FN failed to enhance phytohemagglutinin or OKT-3-induced lymphocyte proliferation and had no effect on CML activity. Moreover, FN did not show mitogenic activity in 3-6-day cultures. Our results demonstrate that FN in vitro is capable of restoring defective lymphocyte proliferation in marrow grafted patients, and circumstantial evidence suggests that this effect is mediated by an interaction between FN and monocytes.

Cellular interactions in marrow-grafted patients. III. Normal interleukin 1 and defective interleukin 2 production in short-term patients and in those with chronic graft-versus-host disease.

Peripheral blood mononuclear leukocytes (cells of marrow donor origin) from 89 patients were collected at various times after allogeneic marrow transplantation, stimulated in vitro by phytohemagglutinin, and assayed for the production of interleukin 2 (IL-2). This was done by testing culture supernatants for their ability to induce proliferation of human lymphoblasts and/or IL-2-dependent cultured murine cytotoxic cells. Supernatants from cultures of patient cells were compared with those of marrow donor cells. Supernatants produced by cells from most short-term marrow recipients (30-101 days postgrafting), regardless of the presence or absence of acute graft-versus-host disease (GVHD), and those from most long-term patients with chronic GVHD (103-1932 days postgrafting) had significantly lower-than-normal IL-2 activity, whereas cells from most long-term marrow recipients without GVHD (353-1934 days postgrafting) had essentially normal IL-2 activity. Additionally, we tested the ability of monocytes from 35 marrow recipients to produce interleukin 1 (IL-1) in response to lipopolysaccharide as compared with monocytes from marrow donors or normal unrelated individuals. IL-1 activity in culture supernatants of patient cells, regardless of the time of testing after marrow grafting and the status of GVHD, was found not to differ from that in supernatants of normal cells. These findings suggest that impaired T cell functions seen in some (but not all) marrow recipients are probably not due to IL-1 but to IL-2 deficiency or to the mechanism that causes IL-2 deficiency.

Cellular interactions in marrow-grafted patients. II. Normal monocyte antigen-presenting and defective T-cell-proliferative functions early after grafting and during chronic graft-versus-host disease.

The proliferation of T cells of marrow donor origin in response to Escherichia coli, an ubiquitous antigen, presented by circulating monocytes of marrow donor origin was investigated in 30 human allogeneic marrow transplant recipients. Compared with cells from healthy marrow donors, T cell proliferation was found to be deficient in all recipients studied 36-71 days after grafting, regardless of the presence or absence of acute graft-versus-host disease and in most patients with chronic graft-versus-host disease studied 118-1804 days postgrafting . In contrast, lymphocytes from most long-term patients without chronic graft-versus-host disease studied 363-2673 days had immune reactivity comparable to that of lymphocytes from their marrow donors. Results of cell-mixing experiments showed that (1) monocytes from most marrow recipients were capable of presenting antigens to normal T cells of marrow donors, and (2) T cells from short-term patients and from long-term patients with active chronic graft-versus-host disease were not induced to proliferate by E-coli-pulsed monocytes from the marrow donors. This inability of T cells to proliferate was likely the result of ineffective interactions among T cell subsets.

Specific suppressor cells and immune response to host antigens in long-term human allogeneic marrow recipients: implications for the mechanisms of graft-host tolerance and chronic graft-versus-host disease.

After marrow grafting from HLA-identical siblings, anti-host immune reactivity and suppressor activity were examined in long-term patients with and without C-GVHD. Lymphocytes (of donor origin) from 17 of 32 patients with C-GVHD showed MLR to non-HLA antigens of stored host lymphocytes, and lymphocytes from 8 of 21 patients with C-GVHD exhibited cytotoxicity to host skin fibroblasts. Lymphocytes from only 2 of 15 patients without C-GVHD, however, showed significant MLR to host lymphocytes and none of 16 had cytotoxicity to host fibroblasts. Suppressor activities differed in the two patient groups. Patients without C-GVHD had circulating suppressor cells that specifically inhibited donor MLR to TNP-host lymphocytes but not to TNP-donor, TNP-unrelated and unmodified unrelated lymphocytes. On the other hand, patients with C-GVHD had predominantly suppressor cells that inhibited donor MLR to TNP-unrelated and unmodified unrelated lymphocytes. These findings indicate that anti-host immune reactivity in the absence of specific suppressor cells plays a role in C-GVHD, and that graft-host tolerance in the stable chimeras may be maintained by specific suppressor cells.

Ineffective cellular interaction and interleukin 2 deficiency causing T cell defects in human allogeneic marrow recipients early after grafting and in those with chronic graft-versus-host disease.

Impairment in T cell proliferation in response to E. coli and in CML to unrelated alloantigens was usually observed in patients early after marrow grafting and persisted in long-term patients with chronic GVHD but not in those without chronic GVHD. We analyzed various cellular functions in the pathway of T cell activation and found that in patients with immunodeficiency, (1) their M phi usually could process and present antigens to normal T cells, (2) their T cells did not proliferate even in the presence of normal antigen-pulsed M phi, (3) IL-2 production by T cells was deficient, and (4) exogenous IL-2 restored CML activity in cells of most patients early after grafting but not in cells of most patients with chronic GVHD. Therefore, failure to induce proliferation and cytotoxicity in T cells of marrow recipients is not likely due to M phi defects but because of ineffective communication among T cell subsets, probably related to impaired IL-2 production and/or unresponsiveness to IL-2.

Inhibition of prostaglandin E2 restores defective lymphocyte proliferation and cell-mediated lympholysis in recipients after allogeneic marrow grafting.

Prostaglandins are said to influence T and B cell function by inhibiting the generation of interleukin 2 (IL 2) and the formation of suppressor lymphocytes. After bone marrow transplantation, patients usually have a profound immunodeficiency that persists in recipients with chronic graft-v-host disease (GVHD) and generally resolves in long-term survivors without GVHD. In vitro tests of lymphocyte function such as allogeneic mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) have been shown to be impaired in many patients. We postulated that prostaglandin E2 (PGE2) plays a role in the impaired in vitro tests. To test this hypothesis, we studied in vitro tests in the presence of PGE2 antagonists, indomethacin, and anti-PGE2 antiserum with cells from 22 short-term patients (less than 100 days postgrafting) and 32 long-term survivors with or without GVHD. Results show that blockade of PGE2 release by indomethacin and anti-PGE2 significantly (P less than .01) enhanced the MLC (+67%) and the CML responses (+10.5%) of cells from long-term survivors with chronic GVHD but not from those of long-term, stable recipients. No enhancement of MLC and CML activity was observed with cells from donors of long-term recipients. In patients shortly after marrow grafting, enhancement in the MLC was not significant. However, CML activity in this patient group was significantly increased (+12.5% in recipients with no GVHD, 8.5% in those with acute GVHD, P less than .01). Indomethacin also suppressed the activity of nonspecific suppressor cells in patients with chronic GVHD. When cells from patients with chronic GVHD were treated with recombinant IL 2 and IL 2 combined with indomethacin, it was possible to get an additional augmentation of lymphocyte proliferation after the addition of indomethacin to IL 2-treated cultures. Thus it is very likely that PGE2 inhibits T lymphocyte proliferation, not exclusively by inhibition of IL2 production or activity. We conclude that PGE2, among other factors, may play a role in the pathogenesis of the immunodeficiency after transplantation. PGE2 does not act primarily by interfering with IL2 but presumably by inducing a suppressorlike activity.

Marrow origin of canine alveolar macrophages.

Pulmonary macrophages were obtained from a canine radiation chimera 221 days after irradiation. A variant of soluble glutamic oxaloacetic transaminase was utilized to demonstrate that these cells were of donor origin, providing evidence that pulmonary macrophages in the dog are of marrow origin.