Prevention of syngeneic graft-versus-host disease by recovery of thymic microenvironment after cyclosporine

Following a course of cyclosporine, syngeneic rat radiation chimeras consistently develop a GVHD-like syndrome. Correlation of the thymic immunopathology with conditions leading to syngeneic graft-versus-host disease (sGVHD) suggested the hypothesis that reconstitution of the normal thymic microenvironment after CsA is necessary for self-tolerance. When thymic regeneration is impaired, as in rats receiving previous mediastinal irradiation, then self-reactive effector cells are not regulated and proceed to damage the target tissues. Alternately, it could be argued that the observed thymic abnormalities are irrelevant to sGVHD. To test the primary hypothesis, post-CsA thymic reconstitution was prevented by total thymectomy in unirradiated rats. These rats consistently developed acute type sGVHD seen at 7 and 21 days post-CsA while rats from the CsA-treated sham thymectomy control group failed to develop sGVHD. Because thymectomy prior to CsA blocks sGVHD, most likely the peripheral effector cells in the post-CsA thymectomy group were derived from the CsA-altered thymus. The absence of sGVHD in the sham group indicates that the thymus led to active regulation of these cells after stopping CsA. If regeneration of the thymus restored only negative selection, then the sham thymectomy group should have also developed sGVHD. Flow cytometry and morphology of the spleen and lymph nodes demonstrated that the thymectomized rats, like CsA-treated radiation chimeras, experienced a significant delay in maturation of T cells following CsA. In contrast to the usual model in radiation chimeras, however, the post-CsA thymectomized rats did not convert to chronic type sGVHD. The importance of an abnormal thymus for this transition was confirmed in syngeneic radiation chimeras. Thymectomy after CsA in these rats also blocked the rapid transition to chronic sGVHD.     

Transfer of cyclosporine-associated syngeneic graft-versus-host disease by thymocytes. Resemblance to chronic graft-versus-host disease

Syngeneic rat radiation chimeras treated transiently with cyclosporine (CsA) often develop a GVHD-like syndrome after discontinuing the drug. CsA also causes medullary involution and loss of medullary epithelium in the thymus. Chronic graft-versus-host disease (GVHD), a late occurring syndrome following bone marrow transplantation with many features of autoimmune diseases, is thought by many to result from a thymic deficiency leading to a failure to develop specific tolerance for the host. A direct connection between a thymic deficiency and chronic GVHD was tested by transferring thymocytes from CsA-treated syngeneic Lewis chimeras into irradiated Lewis secondary recipients. Nine of 10 of these recipients had evidence of chronic GVHD in skin biopsies taken at 3 weeks posttransplant or in the autopsies at 5 weeks. Changes included characteristic lichen planuslike infiltrates and sclerodermalike changes in the skin, characteristic infiltrates and myositis of the tongue, often chronic hepatitis with bile duct injury, and interstitial and ductal infiltrates in the serous salivary glands. Immunoperoxidase stains of the skin and tongue infiltrates showed a marked predominance of W3/25+:OX8- lymphocytes. The hair follicles had increased expression of Ia antigen. The thymus in the secondary recipient had variable thymocyte reconstitution of the cortex and a mild to marked reduction in the relative size of the medulla. Stains for cytokeratin showed a moderate to marked reduction of cortical epithelium and marked to total loss of the medullary epithelium. These studies demonstrate that the features of post-CsA syngeneic GVHD resembling chronic GVHD result from an abnormal thymic microenvironment. They also provide additional evidence linking a thymus deficiency with chronic GVHD.     

Effect of cyclosporine on secondary cytotoxic T lymphocyte responses

Cyclosporine, a potent immunosuppressive agent, inhibits the development of the cytotoxic response in a secondary mixed lymphocyte reaction (MLR) in a dose-dependent manner. Exogenous lymphokines can partially overcome this inhibition. We present evidence that the CsA-resistant cells detected in these responses represent an activated population of memory CTL precursors that require only lymphokines for clonal growth and that kill targets of the original stimulator type only. Recombinant IL-4, when used at high concentrations, can support the generation of CTL in the presence of CsA during a secondary MLR response. The magnitude of the cytotoxic response though is far below the maximal levels achieved either by saturating quantities of rIL-2 or a combination of subsaturable quantities of rIL-2 and rIL-4.     

T lymphocyte subpopulations in bone-marrow-transplanted patients in relation to graft-versus-host disease and cytomegalovirus-induced infection

T cell subpopulations were studied in peripheral blood from 62 bone marrow recipients using monoclonal antibodies and flow cytometry. Patients with acute graft-versus-host disease (GVHD), patients with chronic GVHD, and patients with cytomegalovirus (CMV) infection had decreased OKT4/8 ratios. The data suggest that in GVHD this was due to reduced absolute numbers of OKT4-positive cells. CMV-infected patients had significantly increased numbers of OKT8-positive cells. Monitoring of T cell subpopulations was not informative for diagnosis or prognosis of acute GVHD in the single patient. CMV infection should be suspected at a sudden increase in the number of OKT8-positive cells.     

Antigen-specific T-lymphocyte responses in acute and chronic syngeneic graft-versus-host disease

Administration of cyclosporine (CyA) following autologous bone marrow transplantation elicits a T-lymphocyte autoaggression syndrome with remarkable similarity to acute and chronic graft-versus-host disease (GVHD). This syndrome termed syngeneic (S) GVHD is mediated by a restricted repertoire of autoreactive T cells that promiscuously recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain (CLIP). The present studies evaluated and compared antigen-specific autoreactive T cells during acute and chronic SGVHD isolated ex vivo with a soluble MHC class II-immunoglobulin (sMHC class II-Ig) molecular construct. Two major subsets were detected that had overlapping specificity recognizing the MHC class II binding domain of CLIP but were differentially dependent on the N- and C-terminal flanking domains of this peptide. Both subsets were detected in acute and chronic SGVHD. Interestingly, the cytokine profiles of the CLIP-reactive T cells closely correlated with the onset and progression of disease. Levels of type 1 cytokines, particularly IFN-gamma mRNA production assessed by quantitative polymerase chain reaction (PCR), were dominant during acute SGVHD, whereas chronic SGVHD was associated with type 2 cytokine mRNA production. Although there was a dramatic polarization with respect to cytokine production, only subtle changes in antigen specificity were detected. Of additional interest, autoreactive T cells producing IL-10 mRNA were detected in both acute and chronic SGVHD, suggesting that this cytokine may play an important but perhaps paradoxical role in both the onset and progression of this experimental autoaggression syndrome.     

Chronic graft-versus-host disease in rats after syngeneic bone marrow transplantation

A disease similar to the chronic graft-versus-host disease (cGVHD) seen following transplantation of human bone marrow was observed after syngeneic and allogeneic bone marrow transplantation in rats. Bone marrow grafts were exchanged between donors and recipients that were syngeneic or genetically different for the RT2 erythrocyte antigen locus by the use of AUG/AUG.2B and PVG/PVG.2A congenic pair donor/recipient strain combinations. After an initial period of well-being (120-180 days posttransplantation), several AUG and AUG.2B recipients of syngeneic or RT2-mismatched bone marrow developed clinical signs compatible with cGVHD. The clinical signs of the disease included: erythema, diffuse alopecia, thickened skin folds, and conjunctivitis. Laboratory findings included peripheral blood eosinophilia and impaired in-vitro proliferative responses to third-party spleen cells in the mixed lymphocyte reaction. Histological examination of the tissues of a limited number of rats with cGVHD showed subepidermal mononuclear inflammation with atrophy of the epidermis and adnexa of the skin, as well as plasmacytic hyperplasia of the lymphoid tissues. None of the PVG or PVG.2A recipients of syngeneic or RT2-mismatched marrow developed cGVHD. The development of cGVHD in AUG.2B recipients of syngeneic marrow and the absence of the disease in reciprocal marrow grafts between the PVG/PVG.2A rat strains suggests that the development of the disease in the AUG and AUG.2B recipients of RT2-mismatched bone marrow is not due to the RT2 disparity, but may be due to an autologous immune reaction. Furthermore, the finding that the cGVHD is only observed when the AUG and AUG.2B strains are used as recipients--not when the PVG or PVG.2A strains are used as recipients--suggests that the development of the disease is associated with the genetic background of the host and is independent of the background of the donor. It is possible that the use of high-dose cyclophosphamide treatment is involved in the pathogenesis of cGVHD, because the disease is observed only when the recipients are conditioned for transplantation with this immunosuppressive agent.     

Immunologic reactivity in marrow graft recipients receiving cyclosporine to prevent graft-versus-host disease

In vitro and in vivo immunologic parameters were determined in 26 patients treated continuously with cyclosporine to prevent graft-versus-host-disease (GVHD) after allogeneic bone marrow transplantation (BMT) for acute and chronic leukemia and for aplastic anemia. A group of 18 patients was tested 6 months after BMT and another group of 10 patients was tested after one year. At 6 months after BMT, 94% of the patients had normal serum IgG and IgM levels, whereas at one year 29% of them had low IgA levels. The proportion of patients with normal lymphocyte responses in vitro at 6 months after BMT was 69% and 76% for the responses to concanavalin A and to soluble antigens; 75% and 53% for the responses to allogeneic cells and pokeweed mitogen, respectively; and 89% for the response to phytohemagglutinin. All but one were able to generate suppressor cells upon con A stimulation. At one year after the graft, only one patient had demonstrable multiple abnormalities in in vitro tests. Skin test reactivity at one year was comparable to pre-graft reactivity. After BMT a lymphopenia persisted for 6 months. The rate of infectious complications was high during the first 3 months after BMT, and it diminished progressively as immune functions returned to normal. Infection was the cause of death in two cases (one disseminated cytomegalovirus infection and one septicemia). GHVD occurred in 12 patients; in nine of them the disease was transient and mild, only 1 patient developed severe chronic GVHD. Acute GVHD did not influence the tempo of immunologic reconstitution. In comparison to other studies, it seems that cyclosporine does not delay immune restoration, or increase morbidity from infection, while preventing GVHD and its complications efficiently.     

Induction of murine syngeneic graft-versus-host disease by cells of recipient origin

BACKGROUND: Syngeneic graft-versus-host disease (SGVHD) develops after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a 21-day course of the immunosuppressant cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks after CsA treatment, with inflammation in the colon and liver. It has been demonstrated that CD4+ T cells and a T helper cell type 1 cytokine response (Th1) are involved in the development of SGVHD associated intestinal inflammation. The immune response associated with SGVHD is thought to be the result of the reconstitution of the recipient immune system with the syngeneic donor BM. However, definitive studies have not addressed this issue experimentally. METHODS: To determine the origin of the effector cells that participate in SGVHD, C3H/HeN recipient mice were lethally irradiated and transplanted with BM from normal immunocompetent mice or from immunodeficient, severe combined immune deficient, or Rag-2-/- animals. RESULTS: CsA-treated animals, but not control animals, developed inflammation characteristic of SGVHD in the colon and liver regardless of the source of the donor marrow. Furthermore, immunologically, all CsA treated animals responded similarly with increased production of inflammatory cytokines and an increase in activated CD4+ T cells in the periphery and colon relative to controls. CONCLUSION: These results demonstrate that after lethal irradiation and in the absence of donor T cells, T cells of recipient origin can expand and mediate the induction of CsA-induced SGVHD.     

Cytotoxic T lymphocyte antigen 4-immunoglobulin fusion protein combined with methotrexate/cyclosporine as graft-versus-host disease prevention in a canine dog leukocyte antigen-nonidentical marrow transplant model

BACKGROUND: We studied whether blocking of the T cell costimulatory signal from B7-->CD28 by cytotoxic T lymphocyte antigen 4-immunoglobulin fusion protein would, either by itself or when added to methotrexate/cyclosporine, result in improved graft-versus-host disease prevention after dog leukocyte antigen nonidentical canine hematopoietic stem cell transplantation after 920 cGy total body irradiation. RESULTS AND CONCLUSIONS: Survivals of cytotoxic T lymphocyte antigen 4-immunoglobulin fusion protein-treated dogs were only slightly prolonged over controls. It appeared that the addition of cytotoxic T lymphocyte antigen 4-immunoglobulin fusion protein failed to induce graft-host tolerance in this model beyond that achieved with methotrexate/cyclosporine alone.     

Ruxolitinib on acute graft-versus-host disease prophylaxis after modified donor lymphocyte infusion

ObjectiveTo evaluate the effectiveness of ruxolitinib on acute graft-versus-host disease (aGVHD) prophylaxis and its impact on graft-versus-leukemia (GVL) effect in patients after modified donor lymphocyte infusion (mDLI).MethodsWe retrospectively included patients with relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) who received ruxolitinib prophylaxis between October 2018 and April 2020. The incidence of aGVHD, disease-free survival (DFS), overall survival (OS), and treatment safety were evaluated.ResultsSeventeen patients were followed up for a median time of 8 months (range: 1–26 months). The incidence of aGVHD on Day 30 after mDLI was 41.2% and ranged from Grade 1 to 4; ten of 17 patients (58.8%) achieved a complete response (CR), and two (11.8%) had a partial response (PR). Cytomegalovirus (CMV) reactivation rate was 23.5%, and the median time from mDLI to CMV reactivation was 48.5 days. The mean DFS and OS after mDLI were 1.0 (95% CI 0.0–3.5) and 9.0 (95% CI 1.2–16.8) months, respectively. The causes of death for 10 patients were leukemia relapse (n = 5), aGVHD and septic shock (n = 3), intracranial lesion (n = 1), and COVID-19 (n = 1).ConclusionsWe reported encouraging results of ruxolitinib monotherapy in the prevention of aGVHD and maintenance of GVL for post-transplantation relapsed patients, even though being at high risk with poor initial prognosis.     

Timing of lymphocyte transfusion and portal clamping for the development of lethal graft-versus-host disease in the rat

8 or 1.5 × 10⁸ cells/350 g body weight were injected via the tail vein into (LEW × BN)F₁ rats. The injection of 4 × 10⁸ of LEW rat splenocytes induced lethal GVHD in all nontreated F₁ rats, while 1.5 × 10⁸ of LEW splenocytes did not induce any signs of GVHD. However, when the F₁ rats had received the same dose of parental LEW lymphocytes in combination with portal clamping, 14 recipients out of the 15 rats (93%) suffered GVHD and 10 rats (67%) died from GVHD. Interestingly, portal clamping 7 days after the injection enhanced the incidence of GVHD, whereas no GVHD was observed in the intervention group either 3 or 7 days prior to the cell transfusion. When 1.5 × 10⁸ of allogeneic lymphocytes were injected intravenously togather with 0.1–1.0 mg/kg of endotoxin instead of portal clamping, the injection led the early death by GVHD, while the injection of methylpredonisolone did not enhance GVHD. These results thus indicate that either simultaneous or delayed surgical intervention has the potential to trigger a dormant state in transferred alloreactive lymphocytes. (Received for publication on June 9, 1997; accepted on Jan. 6, 1998)     

Antigen-pulsed neutrophils bearing Ia antigens can induce T lymphocyte proliferative response to the syngeneic or semisyngeneic antigen-primed T lymphocytes

Antigen-pulsed neutrophils from mouse peritoneal cavities displayed a remarkable level of lymphocyte proliferative activities to antigen-primed T lymphocytes. Genetic mapping studies demonstrated that compatibility at the I-A, as well as I-E/C, subregions of the major histocompatibility complex (MHC) was essential for effective presentation of the lysozyme antigen. These antigen-presenting activities were remarkably inhibited by anti-Ia sera. Inhibition tests revealed that neutrophil immune-associated (Ia) antigens seem to be essential for antigen presentation during the initial 8 hr. Elimination studies of antigen-pulsed neutrophils with alloantisera plus complement revealed these antigen-presenting neutrophils bearing both I-A and I-E/C gene products on the same cells. These results suggest that Ia-positive neutrophils might play a role in the immune response through antigen presentation.     

In vitro generation of helper T cells and suppressor T cells that regulate the cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells

Helper T cells and suppressor T cells have been generated in vitro that regulate the cytolytic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified syngeneic cells. B6D2F1 helper cells generated to TNP-modified parental (P1) cells augment the CTL response to those P1-TNP-modified antigens but not to P2-TNP-modified antigens. The generation of these helper T cells requires the presence of splenic adherent cells and these helper T cells are radioresistant. A soluble factor can be obtained from the helper T cell cultures that can also augment the CTL response. The suppressor T cells generated in culture do not demonstrate the specificity observed with the helper T cells; however, they are antigen-dependent in their induction. Whether helper or suppressor activity is obtained depends upon the length of time cells are cultured in vitro.