Immunocompetence of lymphocytes from pregnant mice studied in graft versus host reaction

The ability of lymphocytes taken during the second trimester from C57BL/6 mice mated with CBA males to induce the graft versus host reaction in (CBAxC57BL/6)F₁ hybrids was weaker than that of cells both of virgin donors and of mice pregnant after syngeneic mating. This was reflected in lengthening of the life span of the experimental recipients and weakening of inhibition of endogenous colony formation in the spleen of sublethally irradiated hybrids. This ability was restored at the end of pregnancy and in some experiments it actually exceeded the control.Department of Microbiology, Smolensk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 310–312, March, 1977.     

Influence of lipopolysaccharide on graft versus host reactivity of lipopolysaccharide-unresponsive C3H/HeJ mice.

It was initially reported that lipopolysaccharide (LPS)-unresponsive C3H/HeJ mice are refractory to LPS at the B-lymphocyte level, but more recently it has been shown that other cells are similarly unaffected. The current study was undertaken to study an in vivo LPS-modulated disease process involving macrophage-T cell interactions. Adult CBA/J and C3H/HeJ mice were used as spleen donors, and graft versus host reactions were induced in BALB/c neonates. Prior LPS treatment of CBA/J adults decreased the ability of their spleen cells to cause fatal graft versus host disease in BALB/c neonates, whereas no difference was found between injection of spleen cells from normal or LPS-treated C3H/HeJ mice. Similar results were obtained with these cell types when the mouse spleen mixed leukocyte culture system was used. In a carbon clearance assay for stimulation of the reticuloendothelial system with LPS, it was found that the rate of phagocytosis was significantly increased in BALB/c and CBA/J mice 72 h after inoculation of LPS. No stimulation was seen in rate of carbon uptake in the C3H/HeJ animals after treatment with phenol-extracted LPS or with butanol-extracted LPS. An LPS-induced protective serum factor was produced only in the LPS-responsive CBA/J mice and was specific for the syngeneic cells.     

Intestinal graft versus host disease.

An ileocolectomy specimen was examined from a patient with graft versus host disease (GvHD). In addition to the characteristic histological features of this condition, both the small and the large intestine showed extensive destruction of mucosal tissue with survival of clusters of enterochromaffin cells. This appearance has previously been described only in the large bowel. Endocrine cells seem to be less vulnerable to the effects of GvHD than epithelial cells, resulting in their being spared, which is not seen in other types of crypt destruction.     

Autoreactive B-cell repertoire in mice with chronic graft versus host disease

A quantitative analysis of the frequencies of autoantibody producing B-cells has been undertaken by producing and analyzing random hybridoma collections generated in fusions with activated B-cells. Activated B-cells were derived from mice injected with LPS and SRBC and normal mice. They were compared to those derived from mice undergoing chronic GVHD. The frequencies of successful fusion events correlate well with the number of activated B-cells used in the fusions, so that it is reasonable to conclude that the hybridoma collections reflect the activated B-cell repertoires in the different animals. The frequencies of hybridomas producing autoantibodies as well as their specificities for self-antigens, were not significantly different between the different collections of hybridomas. Moreover, no difference in VH gene family expression was found in the different collections of autoantibody producing hybridomas. So, the activated autoreactive B-cell repertoires in GVHF1 mice and in normal mice is similar. In contrast to the normal activated autoreactive B-cell repertoires, which make predominantly IgM antibodies, the GVH-activated autoreactive B-cells make predominantly antibodies of the IgG class. Therefore, we conclude that T-cell mediated graft versus host activation does not generally lead to selective expansion of autoreactive B-cells, but appears to play a crucial role in the switch from IgM to IgG production.     

The effect of age, sex and breeding on graft versus host reactivity of spleen cells from C57BL mice.

Spleen cell graft versus host (GvH) reactivity was determined in male and female, either virgin or breeder, C57BL mice from 3 to 24 months of age. The GvH reaction was assessed by a local popliteal lymph node assay and by a splenomegaly test for a systemic reaction. Although the GvH reactivity declines progressively with age in both sexes the virgin female response was greater than that of males throughout the period of 6-18 months of age. Two-year-old mice of both sexes were practically unable to mount a GvH reaction. No differences were evident in aging female reactivity after one or two syngeneic pregnancies. On the other hand, 3 or more consecutive pregnancies resulted in enhanced GvH reactivity of 12-24-month-old females, which responded comparably to young virgin mice. This long-lasting immunopotentiating effect of multiparity was similar after 3-4 and 8-9 pregnancies. The possible role of developing foetuses on the maintenance of high GvH reactivity in breeder females is suggested.     

Immunoglobulins and antibody formation in mice during the graft versus host reaction

During the GVH reaction produced in adult (C57B1 ☿×CBA ♂) F₁ hybrid mice by injection of C57B1 cells a profound drop in serum immunoglobulin levels was observed. The fall in IgG and IgA values was not reversed by antigenic stimulation with BSA, but the IgM level rose somewhat after BSA though never to the level seen in immunized controls. The antibody response to BSA, as measured by passive haemagglutination, was grossly depressed in experimental animals. Animals undergoing the GVH reaction showed less local inflammatory response at the site of Freund''s adjuvant injection than did controls. The possible importance of this immunoglobulin deficiency in the clinical presentation of the GVH syndrome is considered.     

The sweat gland in graft versus host disease.

Sweat gland abnormalities occur much more frequently than hitherto described in cutaneous graft versus host disease (GVHD). Two patterns of abnormalities were identified in 80 per cent of cases of acute GVHD: a cytopathic pattern consisting of a combination of basal vacuolopathy with or without lymphocytic infiltration and basal cell degeneration, and a proliferative pattern consisting of basal cell hyperplasia. In chronic GVHD, complete sweat gland destruction with fibrosis was commonly observed. Squamous metaplasia and dilation of the sweat glands were less frequently identified. Ki67 immunostaining confirmed proliferative activity in the basal cells of the distal duct. HLA-DR antigens were expressed on the basal cells of the duct and secretory glands in acute GVHD but not in normal skin. Langerhans cells were absent in both normal and abnormal sweat glands. The role of HLA-DR or Langerhans cells in the initiation of GVHD is questioned in the light of the new data and the primary involvement of proliferating cells is confirmed.     

Antigenaemia during acute graft versus host disease.

AIMS--Animal studies have shown that antigens present within the gut play an important role in the development of acute graft versus host disease (GvHD) following allogeneic bone marrow transplantation (BMT). In previous studies, inert sugars have been found to penetrate the small bowel mucosa after conditioning therapy for BMT; endotoxaemia can also occur during acute GvHD. Data on absorption of antigenic proteins across the gut following BMT in humans have not been presented as yet. METHODS--Six patients undergoing allogeneic BMT were studied to determine whether enteric ovalbumin absorption increased or endotoxaemia developed during acute GvHD. RESULTS--Three patients had minimal antigenaemia and no detectable endotoxaemia before receiving conditioning therapy. At the onset of acute GvHD, however, much higher ovalbumin concentrations were detected in those patients with severe antigenaemia. Serum concentrations of specific antiovalbumin IgG and IgA, or antiendotoxin IgM or IgG had no bearing on detectable IgG or IgM ovalbumin or endotoxin concentrations. In five of six patients, small bowel permeability increased, as tested by the lactulose/mannitol sugar absorption test, but detectable ovalbumin absorption increased in only three of these and only two developed endotoxaemia. CONCLUSIONS--Antigens present within the gut can cross the mucosal epithelium during acute GvHD, probably resulting in an enhanced immune response.     

A study of the lymph-node type of graft versus host reaction in mice

A local form of the graph versus host reaction (GVHR) was induced in adult (CBA×C57BL)F₁ hybrid mice by subcutaneous injection of semiallogeneic spleen, thymus, or bone marrow cells from CBA mice into the right hind footpad. The criteria of activity of the GVHR were an increase in the number of blast forms in the region of popliteal lymph node and in its weight 7 days after transplantation of cells. After transplantation of 5×10⁶ and 20×10⁶ spleen cells the absolute weight of the regional lymph node was increased by 3–5 times and was significantly higher than in the control (injection of living syngeneic or fragmented semiallogeneic cells from the same source). By contrast with the control, in the experimental animals the effect clearly depended directly on the dose of transplanted cells. Enlargement of the lymph nodes was accompanied by the regular appearance of blast forms in them. Thymus and bone marrow cells had a much weaker action than spleen cells.Department of General Pathology, Institute of Clinical and Experimental Medicine, Academy of Medical Sciences of the USSR Siberian Division, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 338–339, March, 1976.     

Protection of newborn mice from graft versus host disease by maternal pre-immunization

Alloimmunization of BALB/c (H-2d) female mice with allogeneic spleen cells from C57BL/6 (H-2b) or CBA/H (H-2k) mice protects BALB/c offspring from graft-versus-host disease (GVH-D) following neonatal intraperitoneal inoculation of high doses of spleen cells respectively of C57BL/6 or CBA/H strains of mice. The mice survived GVH-D over one year after the allogeneic inoculum 24-48 h after birth and they did not show any signs of GVH reaction nor splenomegaly. We show that this phenomenon is antibody mediated and affects the developing immune system of the foetus. Repeated immunization of virgin female BALB/c with anti-H-2b or anti-H-2k antisera (Ab1) can equally abrogate GVH-D in their newborn offspring challenged at 24-48 h after birth with allogeneic spleen cells of H-2b or H-2k phenotype. Our results demonstrate that protection from GVH-D is not specific to the immunizing strain and occurs when the neonatal mice are challenged with C57BL/6 or CBA/H spleen cells. There is thus crossreactivity of tolerance against H-2 specificities. In this study we also report on the in vitro cellular immune responses of the surviving GVH-resistant mice and demonstrate that these responses against both the challenge and third party lymphocytes are impaired.     

Local graft versus host reaction in the xenogeneic system.

The injection of viable rat spleen cells beneath the renal capsule of cyclophosphamide (CY)-treated adult mice produced a localized graft versus host reaction (GvHR), manifested by the kidney enlargement index (KI). The optimal xenogeneic GvHR was obtained after injection of 30-50 million rat spleen cells into mouse recipients treated 24 hr before with CY, 200 mg/kg intraperitoneally (i.p.), and killed 7 days later. Splenomegaly in recipient mice suggested systemic dissemination of the local GvHR.     

Increase in severity of graft versus host disease by cytomegalovirus.

An allogeneic transplant recipient developed severe graft versus host disease (GvHD) 48 days after transplantation that was concomitant with a cytomegalovirus (CMV) viraemia, from which she subsequently died. CMV infection was detected in blood by the polymerase chain reaction and later in tissue by immunohistochemical techniques. CMV should be considered in patients in whom GvHD does not respond to appropriate treatment, and this case suggests that herpes viruses may increase the severity of GvHD by synergistically enhancing the graft versus host reaction.     

Diagnostic features of transfusion associated graft versus host disease.

AIMS--To define the immunopathological profile of transfusion associated graft versus host disease (TA-GvHD) to elucidate its pathophysiology and to determine if any features are of diagnostic value. METHODS--Nine patients (age range 14-61 years) who developed histologically confirmed TA-GvHD between 1989 and 1992 were studied. Immunohistochemical analysis of frozen and formalin fixed skin biopsy tissue was performed. Sections were stained with antibodies to CD3, CD8, CD4 and HLA-DR, using a routine streptavidin-biotin technique with standard diaminobenzidine development. RESULTS--All biopsy specimens showed aberrant positive expression of HLA-DR by epidermal keratinocytes. In four patients, all of whom died, HLA-DR was diffusely expressed throughout the epidermis; in the other five cases keratinocyte expression of HLA-DR was more focal. In all biopsy specimens T cells had infiltrated the dermis and epidermis. In all nine cases CD4+ T helper/inducer cells were the predominant T cells. DISCUSSION--Immunohistochemical studies are of value in the diagnosis of TA-GvHD. Aberrant keratinocyte expression of HLA-DR and dermal and epidermal infiltration of CD4+ T cells are immunopathological features of TA-GvHD. Immunohistochemical analysis of skin biopsy tissue using antibodies to these markers is thus a useful investigation in pancytopenic patients presenting with unexplained rashes.     

Transfusion associated graft versus host disease in an immunocompetent patient.

Transfusion associated graft versus host disease is a rare disorder usually confined to patients who are immunosuppressed. A case is described in a 77 year old woman who was presumed immunocompetent. She was transfused with one unit of blood from an individual who was homozygous for the same HLA haplotype as her. The diagnosis of transfusion associated graft versus host disease should be suspected in a patient who develops aplastic anaemia within 30 days of a transfusion of blood products. It is suggested that blood donations from first degree relatives should not be permitted, unless the donation is irradiated to prevent lymphocyte proliferation.     

Langerhans' cells are depleted in chronic graft versus host disease.

AIMS: To measure Langerhans' cells in skin of patients treated by bone marrow transplantation who developed chronic graft versus host disease (GvHD); to determine whether the reduction in Langerhans' cells resulted directly from the GvHD or from other factors, such as the immunosuppressive regimens used in bone marrow transplant patients. PATIENTS AND METHODS: Lesional and nonlesional skin specimens from nine patients with lichen planus-like lesions and three patients with sclerodermoid lesions were studied. Control skin specimens were taken from three patients undergoing breast reduction surgery. The number of Langerhans' cells/mm2 and the area of Langerhans' cells as a percentage of total epidermis were measured by counting cells labelled with antihuman CD1a. RESULTS: A significant reduction in Langerhans' cell area and number were found in specimens with lesions (area 3.5%; number 507/mm2) compared with specimens without lesions (8.42%; 2375/mm2). In contrast, Langerhans' cell area and number in skin without lesions were similar to controls (10.26%; 2968/mm2). CONCLUSIONS: Langerhans' cells were significantly reduced in skin with lesions of chronic GvHD but not in skin without lesions from the same patient, suggesting that the reduction is a direct consequence of GvHD and not linked to immunosuppressive drugs or late effects of conditioning regimens. In long term bone marrow transplant recipients, Langerhans' cells are derived mainly from the donor cells; therefore, this result suggests the occurrence of autoreactive phenomenon in chronic GvHD.