Thymic selection and thymic major histocompatibility complex class II expression are abnormal in mice undergoing graft-versus-host reactions.

The graft-vs.-host reaction (GVHR) results in damage to the epithelial and lymphoid compartments of the thymus and thus in abnormal maturation and function of thymocytes in mice undergoing GVHR. In this report, the effects of GVHR on thymic T cell receptor (TCR) expression and usage have been investigated. GVHR was induced in unirradiated F1 hybrid mice by the intravenous transfer of parental lymphoid cells. Expression of the CD3/TCR complex on thymocyte subsets defined by CD4 and CD8 was studied by three-color flow cytometry. The level of CD3/TCR was decreased on CD4+CD8-, but not CD4-CD8+, mature thymocytes. The lack of upregulation of CD3/TCR on CD4 single-positive thymocytes, but not on their CD8+ counterparts, suggested an abnormality of class II major histocompatibility complex (MHC) expression in the thymuses of mice undergoing GVHR. Immunofluorescence staining of thymic frozen sections revealed that MHC class II expression was dramatically decreased in GVH-reactive mice. GVHR-induced changes in positive and negative selection were evaluated by determining the incidence of specific V beta TCR segment usage in the thymus. In normal mice, thymocyte usage of any given V beta segment was highly consistent between individuals of the same strain and age; however, a marked divergence in the incidence of TCR V beta 6hi and V beta 8hi cells between GVH-reactive littermate mice was observed, suggesting that thymic positive selection had become disregulated in these animals. Furthermore, negative selection was defective; the incidence of phenotypically self-reactive V beta 6hi T cells was significantly greater in the thymuses of GVH-reactive mice bearing the endogenous superantigen Mls-1a than in untreated controls. Thus, mice undergoing GVHR showed defective TCR upregulation on CD4+CD8- thymocytes and changes in TCR usage reflecting aberrant thymic selection, in conjunction with decreased expression of MHC class II. Most abnormalities of TCR expression and usage on CD4+ thymocytes observed in GVH-reactive mice were analogous to those of class II knockout mice.

Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease.

In this report we have investigated macrophage (M phi) activity and tumor necrosis factor alpha (TNF-alpha) production during graft-vs.-host disease (GVHD). TNF-alpha production by M phi requires two signals: priming of M phi by interferon followed by triggering of TNF-alpha production and release by lipopolysaccharide (LPS). The state of M phi activation was examined in nonirradiated B6AF1 recipient mice injected with either 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) parental B6 lymphoid cells. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS-triggered release of significant amounts of TNF-alpha into the serum resulting in death of the animals within 36 h. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-alpha. In vitro studies demonstrated that M phi were primed during GVHD. The level of M phi priming was greater during acute GVHD than nonlethal GVHD since 100-fold less LPS was required to trigger killing of a TNF-alpha-sensitive cell line by M phi from acute GVHD animals. The amount of TNF-alpha released into the serum after LPS injection increased during the course of the GVHD and was significantly greater in acute GVH-reactive mice. Endogenous LPS was detected in the serum of acute GVH-reactive animals coincident with the onset of mortality. The data provide evidence that during GVHD M phi are primed as a result of the allogeneic reaction and that endogenous LPS therefore triggers M phi production of TNF-alpha resulting in the symptoms characteristic of acute GVHD.

Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice.

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

Mammary carcinoma arising in mice undergoing a chronic graft-versus-host reaction.

A graft-versus-host reaction (GVHR) was induced in 30 (CBA X A)F1 mice by the iv injection of 50 X 10(6) parental strain A lymphoid cells. Solid tumors emerged in 5 of 10 experimental animals that survived beyond 14 months after the GVHR was initiated. The neoplasms were judged to be mammary carcinomas by light and electron microscopic examinations. C-type RNA viral structures were observed in some tumor cells. The neoplasms were successfully transplanted into syngeneic animals by either sc or ip injections of tumor cell suspensions. Tumor transfer to syngeneic mice was not possible if only spleen cells from tumor-bearing animals were transferred. No tumors developed in an age-matched control group that received no treatment.

Experimental stimulation of cell-mediated immunity without concomitant stimulation of humoral immunity in graft-versus-host immunosuppressed mice.

The immunosuppressive effect of the graft-versus-host (GVH) reaction was studied in CBA X A F1 (CAF1) mice which had been rendered immunologically unresponsive by the injection of parental A strain lymphoid cells (GVH mice). Suppression of both cell-mediated and humoral immune responses was demonstrated by the prolonged survival of C57BL/6 (B6) skin allografts and by the inability of GVH mice to produce detectable antibody following stimulation with sheep erythrocytes (SRBC). Appropriate stimulation of GVH mice induced cell-mediated immune reactions to xeno- and allogeneic antigens while the humoral immune responses to the same antigens remained suppressed. Multiple challenges of the GVH mice with B6 tissue caused a rapid rejection of subsequent B6 skin grafts but failed to stimulate the production of any detectable antibodies to B6 allotransplantation antigens. Sensitization of GVH mice with SRBC in Freund's complete adjuvant stimulated a delayed hypersensitivity response to SRBC, although no humoral response to SRBC could be detected, even after three challenges with SRBC. The experimental results are discussed in terms of a proposed model for GVH-induced immunosuppression.

The effect of the graft-versus-host reaction on B lymphocyte production in bone marrow of mice. Depressed genesis of early progenitors prior to mu heavy chain expression.

The effects of systemic graft-versus-host (GVH) reactions on the early precursor cell populations involved in primary B lymphocyte genesis have been examined in the bone marrow of (C57BL/6xA)F1 mice injected with lymphoid cells from A strain mice. Double immunofluorescence labeling techniques for the intranuclear enzyme, terminal deoxynucleotidyl transferase (TdT), the B220 cell surface glycoprotein detected by monoclonal antibody, 14.8, and surface or cytoplasmic mu chains of IgM (s mu, c mu) were used to quantitate 3 putative early B lineage progenitors preceding mu chain expression (TdT+14.8-mu-, TdT+14.8+mu- and TdT-14.8+mu-), pre-B cells (c mu+, s mu-) and B lymphocytes (s mu+). After initiating GVH reactions, the early B precursor cells, pre-B cells, and B lymphocytes in the bone marrow all fell rapidly in numbers, being almost completely absent from 10-15 days to the end of the 30-day assay period. The decline of some of the early progenitors started at a later time and was less complete than that of the more differentiated B lineage cells. In the spleen, B lymphocytes declined rapidly in numbers after 8 days to less than 5% of normal values from 12 days onward. The results demonstrate that systemic GVH reactions in mice almost completely eliminate the B cell lineage, including early precursor cells apparently undergoing mu chain rearrangement in the bone marrow. The pattern of depletion suggests that a range of B lineage progenitor cells may be directly susceptible to GVH reactions. The findings contribute to a model for the pathogenesis of the humoral immunodeficiency of systemic GVH disease.

The graft-versus-host reaction and immune function. III. Functional pre-T cells in the bone marrow of graft-versus-host-reactive mice displaying T cell immunodeficiency.

Studies were performed to determine whether pre-T cells develop normally in the bone marrow of mice displaying thymic dysplasia and T cell immunodeficiency as a consequence of a graft-versus-host (GVH) reaction. GVH reactions were induced in CBAxAF1 mice by the injection of A strain lymphoid cells. To test for the presence of pre-T cells in GVH-reactive mice, bone marrow from GVH-reactive mice (GVHBM) was injected into irradiated syngeneic F1 mice and 30-40 days later thymic morphology and function were studied. Morphology studies showed nearly normal thymic architectural restoration; moreover, such glands contained normal numbers of Thy-1-positive cells. Functional pre-T cells were evaluated by transferring thymocytes from the irradiated GVHBM-reconstituted mice into T-cell-deprived mice. These thymocytes reconstituted allograft reactivity, T helper cell function and Con A and PHA mitogen responses of T-cell-deprived mice. These results suggest that the pre-T cell population in the bone marrow is not affected by the GVH reaction. Therefore, the T cell immunodeficiency associated with the GVH reaction is not due to a deficiency of pre-T cells in the bone marrow but is more likely associated with GVH-induced thymic dysplasia.

Prevention of murine graft-versus-host disease by inducing and eliminating ASGM1+ cells of donor origin.

In this study anti-asialo GM1 antibodies (anti-ASGM1) were used to further characterize the effector cells responsible for graft-versus-host (GVH)-induced histopathological lesions: Two different types of ASGM1+ cells were identified: an endogenous ASGM1+ population and an induced ASGM1+ population. Both of the ASGM1+ cell populations exhibited natural killer (NK) cell activity, as assessed by their ability to lyse YAC tumor targets in vitro. Donor C57BL/6 (B6) mice were treated in vivo with anti-ASGM1 to eliminate endogenous ASGM1+ cells. ASGM1+ cells were induced in B6 donor mice by treating the animals with 15 x 10(6) B6 x AF1 (B6AF1) lymphoid cells for 44-48 hr. The induced ASGM1+ cells were eliminated by in vivo treatment with anti-ASGM1. GVH reactions were induced by injecting B6 lymphoid cells into B6AF1 mice. Prior to GVH induction the B6 donor cells were tested for NK cell activity against YAC tumor target cells in vitro and for T and B cell functions by mitogen responses in vitro. GVH reactions were determined by splenomegaly, suppression of the plaque-forming cell (PFC) response to sheep red blood cells (SRBC), suppression of the T and B cell mitogen responses, and the development of GVH-associated histopathological alterations in the thymus, liver, and pancreas. Donor lymphoid cells depleted of endogenous ASGM1+ cells were effective at inducing splenomegaly, severe suppression of immune functions, and histopathological lesions. Donor lymphoid cells depleted of both the endogenous and induced ASGM1+ cells displayed normal T cell mitogen responses and were capable of inducing splenomegaly and partial suppression of the PFC response to SRBC when injected into B6AF1 recipients, however, these lymphoid cells failed to induce both GVH-associated histopathological lesions and severe suppression of T and B cell mitogen responses. These results suggest that semiallogeneic stimulation induces an ASGM1+ population in the donor inoculum that displays NK cell-like function (YAC killing) and that plays a crucial role in inducing GVH-mediated histopathological lesions and severe immunosuppression of both T and B cell responses.

The graft-versus-host reaction and immune function. IV. B cell functional defect associated with a depletion of splenic colony-forming units in marrow of graft-versus-host-reactive mice.

Studies were conducted to determine whether a functional B cell defect occurred in the bone marrow of mice experiencing a GVH reaction (GVHBM). GVH reactions were induced in AxCBA F1 adult mice by an injection of A strain lymphoid cells. The GVH reaction was confirmed by immunosuppression and thymus histology. At various intervals after GVH induction, GVHBM was tested for its ability to restore B cell function in adult thymectomized irradiated mice reconstituted with normal thymocytes. GVHBM cells obtained seven days after GVH induction restored but slightly the plaque forming cell (PFC) response to sheep erythrocytes and the mitogen response to lipopolysaccharide (LPS). GVHBM cells obtained 10 days or later failed to reconstitute the PFC or LPS responses. GVHBM cells suppressed neither the T or B cell function of normal spleen cells nor the LPS mitogen response of normal bone marrow cells. In addition, the splenic colony-forming units (CFU-s) in GVHBM were slightly decreased by day 10 after GVH induction and markedly depressed by day 22 after GVH induction. These results suggest that the GVH reaction may affect two different events in B cell differentiation. The early decrease in functional B cells that occurs before there is any change in the CFU-s population suggests a direct effect on B cell production, whereas the later absence of functional B cells could be due to the marked decline in stem cell production (CFU-s).

Suppression of B lymphocyte genesis in the bone marrow by systemic graft-versus-host reactions.

The effects of systemic graft-versus-host (GVH) reactions on B lymphocyte production in the bone marrow of mice were examined by quantitating populations of pre-B cells and B lymphocytes. Acute and chronic GVH reactions were induced by injecting A strain lymphoid cells into either (C57BL/6 X A) F1 or (CBA X A) F1 mice, respectively. Control groups of F1 hybrid mice were given syngeneic lymphoid cells. By double immunofluorescence labeling for cytoplasmic mu heavy chains of IgM (c mu) and for surface mu (s mu) the absolute numbers of pre-B cells (c mu + s mu-) and B lymphocytes (s mu +) in the bone marrow and spleen were determined. During acute GVH reactions, the pre-B cells and B lymphocytes in the bone marrow fell rapidly in numbers and were almost absent from 16 days until the end of the 30-day experimental period. In the spleen, the number of B lymphocytes remained normal for 8 days, then fell to less than 2% of control values from 16 days onward. A similar initial decline in pre-B cells and B lymphocytes occurred during chronic GVH reactions. In long-term survivors of GVH reactions, pre-B cells and B lymphocytes began to reappear after 40 days and maintained normal numbers from 100 to 150 days. The antibody response of spleen cells to sheep red blood cells was lost during GVH reactions. However, this occurred even before B lymphocytes were eliminated and the response remained subnormal after B lymphocyte numbers had recovered. The results demonstrate that systemic GVH reactions markedly depress the normally active genesis of primary B lymphocytes in the bone marrow of the host, accounting in part for the associated state of humoral immunodeficiency.

Kinetics of natural killer cell cytotoxicity during the graft-versus-host reaction. Relationship between natural killer cell activity, T and B cell activity, and development of histopathological alterations.

The relationships between splenic natural killer (NK) cell cytotoxicity, T and B cell function, and the development of histopathological lesions in the liver and pancreas have been studied during the course of graft-versus-host (GVH) reactions. GVH reactions were induced in (C57BL/6 X A)F1 (B6AF1) hybrids by different doses, (10,20 and 30 X 10(6)) of either parental strain C57/BL6 (B6) or A lymphoid cells. Splenic NK cell cytotoxicity was studied by employing YAC-1, an NK-cell-sensitive target. Splenic T and B cell function were assessed by mitogen responsiveness to concanavalin A, phytohemagglutinin, and Escherichia coli lipopolysaccharide, and by the in vitro plaque-forming cell response to sheep red blood cells. Histopathological lesions characteristic of GVH reactions were recognized at a time (day 8 after GVH induction) when both T and B cell functions were totally suppressed and NK cell activity was greatest. The severity of histopathological alterations later (day 16 after GVH induction) correlated with an early peak in NK cell cytotoxicity rather than with the overall NK cell activity. When low doses (10,20 X 10(6)) of B6 cells were employed to induce GVH reactions, a significant increase in NK cell activity was observed, yet neither histopathological alterations nor suppression of T and B cell functions were observed. The killing of YAC-1 targets by splenocytes obtained from the different GVH combinations could not be abrogated by pretreatment with anti-Thy 1.2 serum plus complement, suggesting that T lymphocytes were not central to this cytolytic process. These experiments demonstrated that: (1) an inverse relationship between T and B cell function and NK cell activity was observed early after GVH induction, (2) the severity of histopathological lesions and immunosuppression, as well as the degree of overall augmented NK cell activity, was determined by the dose and genotype of donor cells injected to induce GVH reactions, and (3) GVH-associated moderate-severe lesions occurred only in groups in which NK cell activity peaked early--whereas when NK cell activity peaked later, either mild or no lesions were observed, suggesting that the early rapid increase of NK cell activity may be useful for predicting the severity of GVH pathogenesis.

The graft-versus-host reaction and immune function. II. Recruitment of pre-T-cells in vivo by graft-versus-host-induced dysplastic thymuses following irradiation and bone marrow treatment.

The graft-versus-host (GVH) reaction induces thymic dysplasia and an arrest in T cell differentiation. Studies were performed to test the effect of irradiation and reconstitution with bone marrow on GVH-induced thymic dysplasia and T cell differentiation. GVH reactions were induced in CBAxAF1 adult mice by the injection of A strain lymphoid cells. All GVH-reactive mice were immunosuppressed by day 7 after GVH induction and thymic dysplasia was evident by day 24. Forty days after the induction of the GVH reaction the mice were irradiated (850 rads) and repopulated with 10-15 X 10(6) syngeneic or parental bone marrow cells. Thirty days after irradiation and bone marrow reconstitution, GVH-reactive mice were used for histological and functional studies. These mice displayed near-normal thymus morphology with scattered epithelial cells in the medulla, and normal numbers of Thy-1-positive cells. Donor cells had totally repopulated thymuses of irradiated bone marrow reconstituted mice by day 19 after irradiation. T helper cell function did not recover in the reconstituted mice. These results suggest that (1) the process responsible for GVH-induced thymic dysplasia is radiosensitive, and (2) the thymus has the potential to regenerate a normal structure, but fails to regain normal function.

The graft-versus-host reaction and immune function. I. T helper cell immunodeficiency associated with graft-versus-host-induced thymic epithelial cell damage.

The injection of parental A strain lymphoid cells into adrenalectomized CBAxA F1 (BAF1) mice induced a chronic graft-versus-host (GVH) reaction resulting in T cell and B cell immunosuppression as well as thymic epithelial cell injury, but not stress-related thymic involution. Thymocytes from BAF1 mice undergoing a GVH reaction were studied for their ability to reconstitute T helper cell (TH) function and phytohemagglutinin (PHA) and concanavalin A (Con A) mitogen responses in thymectomized, irradiated, BAF1 mice reconstituted with normal syngeneic bone marrow (ATxBM). Thymocytes from BAF1 mice early after the induction of a GVH reaction (days 10-12) were as effective as normal thymocytes in reconstituting TH and mitogen responses. Thymocytes from BAF1 mice 40 or more days after the induction of a GVH reaction did not reconstitute either the TH function or PHA and Con A responses in ATxBM mice. The inability to reconstitute ATxBM mice was not due to the presence of suppressor cells contained in the thymocyte inoculum. It is proposed that GVH-induced thymic epithelial cell injury blocks or arrests normal T cell differentiation, resulting in a population of thymocytes that lack the potential to become competent T helper cells or mitogen-responsive cells when transferred into ATxBM mice. This thymic functional defect results in a permanent TH immunodeficiency in mice experiencing a chronic GVH reaction.

T cell functional defect associated with thymid epithelial cell injury induced by a graft-versus-host reaction.

The injection of parental strain cells into adrenalectomized (CBA x A)F1 mice induced a graft-versus-host (GVH) reaction which was morphologically characterized by thymus epithelial cell injury but not stress-related thymic involution. Thymocytes from mice undergoing a GVH reaction were studied for their ability to reconstitute allograft reactivity in thymectomized, irradiated, bone marrow reconstituted (ATxBM) (CBA x A)F1 mice. Thymocytes of mice experiencing a GVH reaction were theta-positive during the course of the reaction, however, by 40 days after GVH induction these thymocytes were unable to reconstitute allograft reactivity to H-2-incompatible skin grafts. It is proposed that GVH-induced thymic epithelial cell injury prevents or arrests normal T cell differentiation, resulting in a population of thymocytes which lack complete functional capability.

The role of endogenous glucocorticoids on host T cell populations in the peripheral lymphoid organs of mice with graft-versus-host disease.

Previously, we demonstrated that immature CD4+8+ and mature CD4+ thymocyte populations were selectively eliminated during murine graft-versus-host disease (GVHD) as a consequence of elevated levels of endogenous glucocorticoids. In this report, we investigated whether the marked reduction of CD4+8+ and CD4+ thymocyte populations would affect host CD4+ and CD8+ T cell populations in the spleens and lymph nodes (LN) of mice undergoing GVHD. GVHD was induced in (C57BL/6 x A)F1 (B6AF1) mice by injecting A strain parental lymphoid cells. Using an antibody against H2Kb antigens, labeled host B6AF1 cells were distinguished from unlabeled donor A cells. Our results demonstrated a marked deficiency of host CD4+ and CD8+ T cells in the spleens and LN of GVHD mice on day 21 after GVHD induction. The severe reduction of host T cell populations in the peripheral lymphoid organs did not appear to result from the elimination of CD4+8+ and CD4+ thymocyte populations. However, adrenalectomy before GVHD induction reversed the severe loss of both host CD4+ and CD8+ T cell populations in the LN of GVHD mice on day 21, whereas cortisone treatment of adrenalectomized (ADX) GVHD mice resulted in reduction of host LN CD4+ and CD8+ T cell populations similar to that observed in non-ADX GVHD animals on day 21. In addition, adrenalectomy markedly improved the proliferative response of LN T cells to mitogens when compared with immunosuppressed T cells from the LN of non-ADX GVHD mice. In contrast, adrenalectomy did not reverse splenic T cell immunosuppression and the marked reduction of splenic host T cell populations during GVHD. These results suggest that high levels of endogenous glucocorticoids during GVHD play a central role in mediating severe deficiency of host T cell populations and inducing severe T cell immunosuppression in the LN, but not in the spleen, of GVHD mice.

T cell subsets in the thymus of graft-versus-host immunosuppressed mice. Sensitivity of the L3T4+Lyt-2- subset to cortisone.

We have investigated the effects of graft-versus-host disease on T cell differentiation in the murine thymus. We previously reported that GVH-induced thymic dysplasia results in a T cell immunodeficiency associated with a lack of IL-2 production. This deficiency in IL-2 production may be the result of a reduction in the number of L3T4+Lyt-2- IL-2-producing cells or of a functional defect in this population. To test these two alternatives, flow cytometry analysis of L3T4 and Lyt-2 antigen expression on thymocytes along with immunofluorescence microscopy were employed to assess T cell phenotypes in thymuses of GVH mice. GVH reactions were induced by injecting 40 x 10(6) C57BL/6 (B6) or A strain lymphoid cells into C57BL/6xAF1 (B6AF1) mice. Thymocyte populations were quantitated on different days after GVH induction. In the normal thymus, the ratio of L3T4/Lyt-2 single positive cells was greater than 2:1. In contrast, such a ratio was less than 1:1 in the atrophic GVH thymus, owing to a selective reduction in the number of L3T4+Lyt-2- cells. Following cortisone treatment the ratio of L3T4/Lyt-2 single positive thymocytes in normal F1 mice was approximately 3:1, whereas in GVH animals this ratio was reversed (1:2). This reversal was due to a selective reduction in the absolute numbers of L3T4+Lyt-2- cells. In adrenalectomized GVH animals, thymic cortical atrophy was prevented and normal ratios of L3T4/Lyt-2 single positive cells were observed. However, when these animals were treated with cortisone, the L3+T4/Lyt-2- population was more sensitive than was the L3T4- Lyt2+ population, thereby resulting in a 1:2 L3T4/Lyt-2 ratio. These results demonstrate that single positive L3T4 cells are present in the murine GVH thymus, yet they have not acquired cortisone resistance, a trait normally attributed to this mature thymic subset. It appears that the GVH dysplastic thymus can support the differentiation of L3T4+Lyt-2- cells--however, such a thymus is unable to confer cortisone resistance upon this population. Consequently, these cells appear to be eliminated when exposed to corticosteroids in peripheral lymphoid tissue.

Graft-versus-host reactions in the beige mouse. An investigation of the role of host and donor natural killer cells in the pathogenesis of graft-versus-host disease.

To investigate the role of natural killer (NK) cells in the induction and pathogenesis of graft-versus-host (GVH) disease, +/beige (+/bg; normal NK cell activity) and beige/beige (bg/bg; deficient NK cell activity) parental C57BL/6 (B6) lymphoid cells were used to induce GVH reactions in either B6 X C3H/Hej +/bg (+/bgF1) or B6 X C3H/HeJF1 bg/bg (bg/bg F1) hybrid mice. When B6 bg/bg parental lymphoid cells (PLC) were injected into bg/bg F1 mice, early splenomegaly, early severe suppression of the plaque-forming cell (PFC) response to sheep red blood cell (SRBC), and only partial suppression of T cell mitogen responses to concanavalin A (Con A) and phytohemagglutin (PHA) were observed on day 12 after GVH induction. In the same GVH combination, slightly augmented NK cytotoxic activity was induced and no GVH-induced moderate-to-severe pathological alterations in the liver and pancreas were observed. When bg/bg PLC were injected into +/bg F1 mice, early splenomegaly and pronounced immunosuppression of the PFC response to SRBC and partial suppression of Con A and PHA responses were observed on day 12 after GVH induction. In this combination (bg/bg----+/bg F1), significant NK cell activity was induced, but no moderate-to-severe histopathological alterations were observed. In contrast, when B6 +/bg PLC were injected into either +/bg F1 or bg/bg F1 hybrids, early splenomegaly, and severe immunosuppression of both the PFC response to SRBC and the T cell mitogen responses to Con A and PHA were observed by day 12--which persisted until day 30 after GVH induction. Furthermore, high NK cell activity was recorded and moderate-to-severe histopathological alterations appeared in both +/bg F1 and bg/bg F1 recipients. These results show that the bg/bg PLC can induce GVH-associated early splenomegaly and immunosuppression of the PFC response to SRBC in both the bg/bg F1 and +/bg F1 hybrids, but that it failed to induce moderate-to-severe histopathological alterations, even though NK cell activity of host origin was activated during GVH reactions. Conversely, when +/bg donor cells were used to induce the GVH reaction, splenomegaly and immunosuppression, as well as moderate-to-severe histopathological lesions were induced. These results suggest that donor NK cells rather than host NK cells play an active role in GVH-associated tissue damage, which in turn contributes to the long-term suppression.

The effects of polyinosinic:polycytidylic acid on the graft-versus-host reaction. III: Increased severity of the reaction with delayed pI:C treatment.

We have been investigating the effects of polyinosinic:polycytidylic acid (pI:C), an interferon inducer, on the graft-versus-host reaction. We have previously shown that pI:C treatment of C57BL/6xAF1 (B6AF1) recipient mice immediately before injection of C57BL/6 (B6) parental lymphocytes inhibited the immuno-suppression and pathological changes normally caused by the GVH reaction, by a mechanism apparently identical to that seen in F1 hybrid resistance (HR) to hematopoietic grafts. We now demonstrate that delaying pI:C treatment by as little as 48 hr produces the opposite effect. Treatment of recipient B6AF1 mice at different days after transfer of parental lymphocytes induced a marked increase in the severity of the GVH reaction, as measured by a decreased plaque-forming cell response to sheep erythrocytes; decreased proliferative response to the T and B cell mitogens PHA, Con A, and LPS; increased pathological changes in both lymphoid and nonlymphoid tissues; and increased GVH-associated mortality. This effect is unrelated to HR, as pI:C was able to augment the severity of the GVH reaction when A strain cells were injected into AxCBAF1 recipients, which do not manifest HR. Early pI:C treatment (1 and 2 days after parental cell transfer) increased the severity of the GVH reaction much more than later pI:C treatment (7 and 8 days after parental cell transfer). This observation, along with the demonstration of altered pathology in GVH mice treated with pI:C, suggests that the effect of pI:C is not mediated through a direct suppressive effect of IF on the cells responding in either the PFC or mitogen assays, but rather by the ability of IF to activate or suppress mechanisms involved in the development of GVH-induced alterations.

Interleukin-1 and interleukin-2 defects associated with murine graft-versus-host-induced immunodeficiency.

In this study we investigated the mechanism, or mechanisms, involved in graft-versus-host (GVH)-induced T cell immunodeficiency. Chronic GVH reactions were induced in normal CBA X A F1 (BAF1) hybrid mice by the injection of parental A strain lymphoid cells. At various times (43-91 days) after GVH induction, the functional status of GVH T cells was assessed using interleukin-1 (IL-1) and interleukin-2 (IL-2) as probes. The response of GVH thymocytes to IL-1 was depressed when compared with normal thymocytes. Although GVH peanut-agglutinin-negative (PNA-) thymocytes did respond to IL-2 alone or IL-2 plus phytohemagglutinin (PHA), this response was significantly lower than the response of PNA- thymocytes from normal mice. In addition, GVH spleen cells failed to produce significant amounts of IL-2 when stimulated with concanavalin A. These results suggest that the long term immunosuppression associated with murine chronic GVH disease is due, at least in part, to a decrease in the responsiveness to IL-1 and IL-2, and to a marked deficiency in IL-2 production.

Macrophage activity in mice undergoing chronic graft-versus-host reactions.

The functional state of the mononuclear phagocyte system has been investigated in mice undergoing chronic graft-versus-host (GVH) reactions (GVHR), initiated by the injection of parental DBA/2 lymphoid cells into (DBA/2 X C57BL/6)F1 hybrid mice. Macrophage function was assessed in vivo by the ability to develop host resistance to infection with Listeria monocytogenes and found to be normal in GVH mice, as measured by the development of resistance during the early phase of natural (macrophage-mediated) resistance to the infection. During the later phase of acquired immunity to listerial infection, GVH mice had lowered resistance, but this was attributed to impaired T cell immunity rather than to defective macrophage function. The inflammatory response to a phlogistic agent, thioglycolate, was also found to be normal in GVH mice, as measured by the accumulation of inflammatory macrophages in the peritoneal cavity. In an in vitro assessment of macrophage function, phagocytosis was found to be enhanced initially (2 weeks following GVHR induction) but it subsequently became depressed for the duration of the study (12 weeks after GVHR induction). Macrophage chemotactic activity was initially normal, then became depressed and remained so for the duration of the study. Thus, despite the profound suppression of specific immunity induced by the GVH reaction and the functional defects of GVH macrophages apparent in vitro, the response of the mononuclear phagocyte system to in vivo stimuli, such as infection or inflammation, is unimpaired in GVH mice.