scholarly journals Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 182-194 ◽  
Author(s):  
DA Vallera ◽  
SF Carroll ◽  
DC Snover ◽  
GJ Carlson ◽  
BR Blazar
Keyword(s):  
Bone Marrow ◽  
Weight Gain ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Major Histocompatibility ◽  
Toxin A ◽  
Host Disease ◽  
Ricin Toxin ◽  
Graft Versus Host ◽  
A Chain

Abstract Graft-versus-host disease (GVHD) was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow and splenocytes into lethally irradiated B10.BR (H-2k) murine recipients. An immunotoxin (IT) composed of a pan T-cell monoclonal antibody called anti-Ly1 (the murine homologue to human anti-CD5) was conjugated to ricin toxin A chain (anti-Ly1-RTA) and used to treat recipient mice. In vitro, IT was as active as free RTA, bound selectively, and inhibited T- cell proliferation even in the absence of potentiators. Mice administered anti-Ly1-RTA in vivo during ongoing GVHD, at a dose of 10 micrograms/d for 5 days, showed lower numbers of splenic Thy1.2+ T cells and significantly improved survival as compared with mice given phosphate-buffered saline (PBS) or irrelevant control RTA IT. Protection was transient because GVHD and weight loss occurred when injections ceased. Survival could not be enhanced by crosslinking RTA30, a low oligosaccharide-containing fraction of purified RTA. Treatment with anti-Ly1-RTA caused a significant elevation in neutrophils, and higher doses were associated with mild hepatotoxicity. In contrast, infusion of identical doses and schedules of another pan T- cell immunotoxin, anti-Thy1.2-RTA, caused a significant decrease in lymphocytes, but not neutrophils; a precipitous increase in weight; a decrease in total plasma protein (TPP); and an increase in pleural and peritoneal effusions reminiscent of vascular leak syndrome (VLS). Although the toxic effects of anti-Thy1.2-RTA were too severe to show a survival advantage in a GVHD model, histopathologic studies showed a definite anti-GVHD effect. The most significant decline in GVHD as compared with the PBS-treated controls was observed in skin, and to a lesser extent, in liver and lung. To investigate the cause of IT toxicity, anti-Thy1.2-RTA was administered intraperitoneally to lethally irradiated B10.BR (H-2k) recipients of syngeneic bone marrow. These recipients showed the same weight gain, hypoproteinuria, and VLS observed in the GVHD model. Death occurred at higher anti-Thy1.2-RTA doses (30 or 50 micrograms/daily injections administered days 8 through 12 posttransplant). Anti-Thy1.2-RTA had a negligible effect on renal function, but histologic studies showed patchy dropout of the renal tubules. Treatment resulted in pulmonary vascular congestion, but there was no pathologic evidence of liver, brain, or colon toxicity. Weight gain was enhanced by irradiation because nonirradiated normal mice did not undergo such a precipitous weight increase.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicity and efficacy of anti-T-cell ricin toxin A chain immunotoxins in a murine model of established graft-versus-host disease induced across the major histocompatibility barrier

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 182-194 ◽  
Author(s):  
DA Vallera ◽  
SF Carroll ◽  
DC Snover ◽  
GJ Carlson ◽  
BR Blazar
Keyword(s):  
Bone Marrow ◽  
Weight Gain ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Major Histocompatibility ◽  
Toxin A ◽  
Host Disease ◽  
Ricin Toxin ◽  
Graft Versus Host ◽  
A Chain

Graft-versus-host disease (GVHD) was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow and splenocytes into lethally irradiated B10.BR (H-2k) murine recipients. An immunotoxin (IT) composed of a pan T-cell monoclonal antibody called anti-Ly1 (the murine homologue to human anti-CD5) was conjugated to ricin toxin A chain (anti-Ly1-RTA) and used to treat recipient mice. In vitro, IT was as active as free RTA, bound selectively, and inhibited T- cell proliferation even in the absence of potentiators. Mice administered anti-Ly1-RTA in vivo during ongoing GVHD, at a dose of 10 micrograms/d for 5 days, showed lower numbers of splenic Thy1.2+ T cells and significantly improved survival as compared with mice given phosphate-buffered saline (PBS) or irrelevant control RTA IT. Protection was transient because GVHD and weight loss occurred when injections ceased. Survival could not be enhanced by crosslinking RTA30, a low oligosaccharide-containing fraction of purified RTA. Treatment with anti-Ly1-RTA caused a significant elevation in neutrophils, and higher doses were associated with mild hepatotoxicity. In contrast, infusion of identical doses and schedules of another pan T- cell immunotoxin, anti-Thy1.2-RTA, caused a significant decrease in lymphocytes, but not neutrophils; a precipitous increase in weight; a decrease in total plasma protein (TPP); and an increase in pleural and peritoneal effusions reminiscent of vascular leak syndrome (VLS). Although the toxic effects of anti-Thy1.2-RTA were too severe to show a survival advantage in a GVHD model, histopathologic studies showed a definite anti-GVHD effect. The most significant decline in GVHD as compared with the PBS-treated controls was observed in skin, and to a lesser extent, in liver and lung. To investigate the cause of IT toxicity, anti-Thy1.2-RTA was administered intraperitoneally to lethally irradiated B10.BR (H-2k) recipients of syngeneic bone marrow. These recipients showed the same weight gain, hypoproteinuria, and VLS observed in the GVHD model. Death occurred at higher anti-Thy1.2-RTA doses (30 or 50 micrograms/daily injections administered days 8 through 12 posttransplant). Anti-Thy1.2-RTA had a negligible effect on renal function, but histologic studies showed patchy dropout of the renal tubules. Treatment resulted in pulmonary vascular congestion, but there was no pathologic evidence of liver, brain, or colon toxicity. Weight gain was enhanced by irradiation because nonirradiated normal mice did not undergo such a precipitous weight increase.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Therapy for ongoing graft-versus-host disease induced across the major or minor histocompatibility barrier in mice with anti-CD3F(ab')2-ricin toxin A chain immunotoxin

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4367-4375 ◽  
Author(s):  
DA Vallera ◽  
PA Taylor ◽  
A Panoskaltsis-Mortari ◽  
BR Blazar
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Toxin A ◽  
Pharmacologic Agent ◽  
Single Strain ◽  
Host Disease ◽  
Ricin Toxin ◽  
Graft Versus Host ◽  
A Chain

A new pharmacologic agent, anti-CD3F(ab')2-ricin toxin A chain (RTA), was synthesized for the purpose of targeting T cells and as a means of treating established graft-versus-host disease (GVHD). The Fc region of anti-CD3 monoclonal antibody (MoAb) was removed to prevent its ability to activate T cells. The resulting F(ab')2 fragments were conjugated to deglycosylated RTA (dgRTA), a catalytic and potent phytotoxin. The resulting immunotoxin (IT) was potent (greater than 95% inhibition) and selective in inhibiting T-cell mitogenesis in vitro. In vivo, the IT depleted 80% of T cells in mice receiving bone marrow (BM) transplants. Transplantation in an aggressive acute GVHD model using C57BL/6 donor cells and H-2 disparate B10.BR recipients resulted in an infiltration of CD3-expressing cells and a median survival time (MST) of 20 to 30 days. A 5-day course of anti-CD3F(ab')2-RTA (30 micrograms/d intraperitoneally) beginning 7 days after GVHD induction was beneficial in treating established GVHD in these mice, as evidenced by significantly prolonged survival (MST, greater than 80 days), superior mean weight values, and improved clinical appearance. Neither intact anti-CD3, unconjugated anti-CD3 F(ab')2 fragments, nor a mixture of anti-CD4 and anti-CD8 MoAbs (which are highly effective in prophylactic models) were as effective. F(ab')2 fragments made from anti-Lyt-1 (the murine homologue of human anti-CD5) linked to RTA were also not effective, despite the fact that both anti-CD3F(ab')2-RTA and anti-Lyt- 1F(ab')2-RTA had similar half-lives of about 9 hours. The IT also increased MST in two aggressive models of GVHD across non-H-2 minor histocompatibility barriers, indicating that the usefulness of anti- CD3F(ab')2-dgRTA is not limited to a single-strain combination. This agent should be further investigated as an alternative to current strategies for treating steroid refractory GVHD.

scholarly journals Pretreatment of murine donor grafts with L-leucyl-L-leucine methyl ester: elimination of graft-versus-host disease without detrimental effects on engraftment

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 798-805 ◽  
Author(s):  
BR Blazar ◽  
DL Thiele ◽  
DA Vallera
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Methyl Ester ◽  
Graft Versus Host Disease ◽  
Host Cells ◽  
Host Disease ◽  
Murine Bone Marrow ◽  
Graft Versus Host

Abstract Incubation of murine bone marrow and splenocytes with the dipeptide methyl ester, L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), which results in the selective depletion of cytotoxic T cells and their precursors, natural killer cells, and monocytes, completely protected 30 recipients of fully allogeneic donor grafts from lethal graft-versus- host disease (GVHD). These results were comparable with those obtained in 30 recipients of anti-Thy 1.2 plus complement (C')-treated donor marrow. However, in contrast to antibody- and C'-dependent T-cell depletion, which reduces the level of donor cell engraftment in our model system, we did not observe such effects using Leu-Leu-OMe marrow pretreatment. As compared with the 24 H-2 typed recipients of anti-Thy 1.2 + C'-treated donor grafts, the 29 H-2 typed recipients of Leu-Leu- OMe-treated donor grafts had significantly (P less than .001) higher percentages of donor cells (mean = 93% v 74%) and significantly (P less than .001) lower percentages of host cells (mean = 6% v 15%) posttransplantation. In vitro limiting dilution assay (LDA) was performed to assess the comparative efficacy of cytolytic T-lymphocyte (CTL) precursor depletion by Leu-Leu-OMe or anti-Thy 1.2 + C' pretreatment. We observed greater levels of CTL precursor depletion in Leu-Leu-OMe treated as compared with anti-Thy 1.2 + C'-treated bone marrow plus spleen cells (BMS) obtained from nontransplanted mice. This suggests that the in vivo results cannot simply be attributed to a less efficacious functional inactivation of cytolytic T-cell precursors by Leu-Leu-OMe treatment as compared with anti-Thy 1.2 + C' treatment. Immunoreconstitution was similar in recipients of Leu-Leu-OMe-treated grafts and anti-Thy 1.2 + C'-treated grafts 100 days posttransplant. In our opinion, Leu-Leu-OMe marrow pretreatment deserves further investigation as a methodology to achieve GVHD prevention without significantly reducing the propensity toward host cell repopulation.

CD6+ Donor Marrow T-Cell Depletion as the Sole Form of Graft-Versus-Host Disease Prophylaxis in Patients Undergoing Allogeneic Bone Marrow Transplant From Unrelated Donors

2001 ◽  
Vol 19 (4) ◽  
pp. 1152-1159 ◽  
Author(s):  
Robert J. Soiffer ◽  
Edie Weller ◽  
Edwin P. Alyea ◽  
Peter Mauch ◽  
Iain L. Webb ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Bone Marrow Transplant ◽  
Graft Versus Host Disease ◽  
Marrow Transplant ◽  
Cell Depletion ◽  
T Cell Depletion ◽  
Host Disease ◽  
Graft Versus Host ◽  
Gvhd Prophylaxis

PURPOSE: The role of donor marrow T-cell depletion (TCD) in preventing graft-versus-host disease (GVHD) after transplantation of unrelated allogeneic marrow remains undefined. Because different TCD methodologies differ in the degree and specificity with which T cells are removed, it is likely that transplant outcomes would depend on which technique is used. Herein, we report results in the first 48 recipients of unrelated marrow using CD6+ TCD as the sole form of GVHD prophylaxis. PATIENTS AND METHODS: Median age of patients was 46 years (20 to 58 years). Donors were matched at A/B HLA loci. Ablation consisted of cyclophosphamide and fractionated total-body irradiation (TBI; 14 Gy). To facilitate engraftment, patients also received 7.5 Gy (22 points) or 4.5 Gy (26 points) of total lymphoid irradiation (TLI) before admission. No additional immune suppressive prophylaxis was administered. Granulocyte colony-stimulating factor was administered daily from day +1 to engraftment. RESULTS: All 48 patients demonstrated neutrophil engraftment. An absolute neutrophil count of 500 × 106/L was achieved at a median of 12 days (range, 9 to 23 days). There were no cases of late graft failure. The number of CD34+ cells infused/kg was associated with speed of platelet and neutrophil recovery. The dose of TLI did not influence engraftment. Grades 2-4 acute GVHD occurred in 42% of patients (95% confidence interval [CI], 0.28 to 0.57). Mortality at day 100 was 19%. There have been only five relapses. Estimated 2-year survival was 44% (95% CI, 0.28 to 0.59) for the entire group, 58% for patients less than 50 years of age. In multivariable analysis, age less than 50 years (P = .002), cytomegalovirus seronegative status (P = .04), and early disease status at bone marrow transplant (P = .05) were associated with superior survival. CONCLUSION: CD6+ TCD does not impede engraftment of unrelated bone marrow after low-dose TLI, cyclophosphamide, and TBI. CD6+ TCD as the sole form of GVHD prophylaxis results in an incidence of GVHD that compares favorably with many adult studies of unrelated transplantation using unmanipulated marrow and immune-suppressive medications, especially in light of the median age of our patients (46 years). Although event-free survival in patients less than 50 years of age is very encouraging, older patients experience frequent transplantation-related complications despite TCD.

scholarly journals Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 3032-3044 ◽  
Author(s):  
X Liu ◽  
V Chesnokova ◽  
SJ Forman ◽  
DJ Diamond
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
T Cell Receptor ◽  
Graft Versus Host Disease ◽  
Cell Receptor ◽  
Supporting Evidence ◽  
Allogeneic Bone ◽  
Host Disease ◽  
Graft Versus Host ◽  
Beta 2

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus- host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.

scholarly journals Patient Bone Marrow Chimerism at Time of Donor Lymphocyte Infusion Predicts Development of Graft Versus Host Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2132-2132
Author(s):  
Eva AS Koster ◽  
Liesbeth C. de Wreede ◽  
Sylwia Wallet-Malicka ◽  
Lisette Bogers ◽  
Peter van Balen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Hazard Ratio ◽  
The State ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells ◽  
Donor Type

Abstract After allogeneic stem cell transplantation (alloSCT), donor T cells targeting patient derived hematopoietic cells can induce a Graft versus Leukemia (GvL) effect preventing relapse. However, targeting of healthy patient tissues can cause Graft versus Host Disease (GvHD). The inflammatory environment induced by pre transplantation conditioning, the number of donor T cells in the graft, genetic disparity between patient and donor and the presentation of allo-antigens by activated patient derived antigen-presenting cells (APC) to donor T cells play a role in the development of GvL and/or GvHD. Donor T cell depletion (TCD) reduces GvHD and GvL. After TCD alloSCT, postponed prophylactic donor lymphocyte infusions (pDLI) are often needed to induce a GvL effect. When using 10/10 matched donors, our first dose of pDLI at six months after TCD alloSCT contained 3.0x10^6 T cells/kg (related donor, RD) or 1.5x10^6 T cells/kg (unrelated donor, UD). We evaluated whether the risk of developing GvHD after DLI is influenced by the donor type, intensity of the conditioning and/or patient bone marrow (BM) chimerism at time of DLI Sixty patients with acute leukemia (52 AML, 8 ALL; median age 57; 27 RD, 33 UD) received pDLI at a median of 6.4 months after TCD alloSCT in the absence of GVHD or relapse. Twenty-four patients received myeloablative (MA) conditioning consisting of cyclophosphamide and TBI. 36 patients received non-myeloablative (NMA) conditioning based on fludarabin and busulphan. TCD was performed by adding 20mg alemtuzumab to the graft. Only MA conditioned patients with an UD (n=12) received post transplantation ciclosporin as GvHD prophylaxis, which was tapered from 1 month after alloSCT. Clinically significant GvHD was defined as need of therapeutic systemic immunosuppression (tIS) for GvHD for at least 2 weeks or until death. Bone Marrow (BM) chimerism was measured prior to DLI. Three categories of patient chimerism levels were defined: no patient derived cells (absent), patient derived cells present, but < 5% (low), or ≥ 5% (high). In case of persisting or increasing patient chimerim after pDLI, a second DLI was given at 3-6 months after the first. A multi-state model was designed (Figure 1) with the first DLI (DLI1) as starting state and time. Patients starting tIS after DLI1 transit to the state tIS. Patients who need a second DLI, develop a relapse or die, transit to these respective states. Patients who stay in the state of DLI1 are considered to have a positive outcome. All patients had a follow-up of at least one year after DLI. Numbers in the boxes in Figure 1 represent the number of patients in that state at 1 year after DLI1 and numbers next to the arrows indicate the numbers of patients who made the transition between the two states. Donor type (unrelated versus related), conditioning (NMA versus MA) and patient BM chimerism at time of DLI were included in a Cox model for the transition hazards to investigate their association with the development of GvHD after DLI. For the total group, the cumulative incidence of tIS at 1 year after pDLI was 33% (95% CI 21-45%). Patients with an UD had a hazard ratio (HR) of 1.1 (95% CI 0.4-3.3) of needing tIS after DLI1 compared to patients with a RD. Compared to MA conditioning, NMA conditioned patients had a hazard ratio of 2.1 (95% CI 0.5-8.9) of needing tIS after DLI. They had a HR of 0.2 (95% CI 0.04-0.95) of stopping tIS compared to MA conditioned patients, indicating that DLI after NMA conditioning is associated with more severe GVHD. We hypothesized that this was due to the persistence of patient derived APC. BM chimerism at time of DLI was measured in 47 patients. After NMA and MA conditioning, BM patient chimerism was absent in 14% and 56%, low in 41% and 39%, and high in 45% and 6%, respectively (Fisher's exact test p=0.002 for difference between type of conditioning). Compared to the group without patient chimerism, the low and high patient chimerism group had a HR of 1.9 (95% CI 0.9-4.2) and 3.6 (95% CI 1.7-8.0) of needing tIS after DLI, respectively (Figure 2), demonstrating that the level of patient chimerism is a strong predictor for development of GvHD after DLI, even when taking into account the type of conditioning regimen. Patient BM chimerism at time of pDLI is a strong and independent predictor for the risk of developing GvHD. Dose reduction in case of an UD equalized the GvHD risk compared to a RD. When choosing a T cell dose for pDLI, patient chimerism should be considered a relevant parameter. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prevention of murine graft-versus-host disease and bone marrow alloengraftment across the major histocompatibility barrier after donor graft preincubation with anti-LFA1 immunotoxin

Blood ◽  
1991 ◽  
Vol 78 (11) ◽  
pp. 3093-3102
Author(s):  
BR Blazar ◽  
SF Carroll ◽  
DA Vallera
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Graft Versus Host Disease ◽  
Cell Activity ◽  
Target Cells ◽  
Major Histocompatibility ◽  
Host Disease ◽  
Graft Versus Host ◽  
Alpha Chain

We have investigated the effects of the in vitro depletion of LFA1 positive cytolytic T lymphocytes, natural killer (NK) cells, and monocytes on the afferent phase of graft-versus-host disease (GVHD). Lethal GVHD was induced across the murine major histocompatibility complex by injecting C57BL/6 (H-2b) bone marrow (BM) cells (a source of stem cells) and splenocytes (S) (a source of T cells) into lethally irradiated B10.BR (H-2k) recipients. Because anti-LFA1 does not bind complement (C′) effectively, we conjugated anti-LFA1 alpha chain monoclonal antibody (MoAb) to ricin toxin A chain (RTA) as a means of facilitating target cell elimination. A 2-hour preincubation of C57BL/6 bone marrow/spleen (BMS) with anti-LFA1-RTA in the presence of ammonium chloride (a potentiator of immunotoxin toxicity), but not a control immunotoxin (IT), reduced CTL activity by greater than 2 logs, significantly reduced NK cell activity, and prevented B10.BR mice from developing GVHD. Depletion of target cells by toxin-labeled-MoAb and not the blockade of the LFA1 molecule by the anti-LFA1 MoAb accounted for our results, because incubating cells with IT in the absence of a potentiator had no effect on GVHD prevention. In contrast, C57BL/6 recipients of C3H BMS grafts only partially benefited from anti-LFA1- RTA preincubation, demonstrating that in this system, different cells not expressing LFA1 were involved in GVHD generation. The same findings observed with anti-LFA1-RTA preincubation were observed with preincubation with L-leucyl-L-leucine methyl ester, a chemical compound eliminating cytolytic cells, providing further support that GVHD induction in the C3H/HeJ into C57BL/6 system is not entirely mediated by classical cytolytic T cells. We next tested anti-LFA1-RTA in a model devised to measure its effect on alloengraftment (B10.BR recipients given lower doses of irradiation). Anti-LFA1-RTA BM preincubation selectively reduced alloengraftment in the model. This observation, combined with experiments showing that LFA1-RTA preincubation, but not anti-Thy 1.2 + C' or control IT preincubation, reduced colony-forming unit-spleen formation, indicates that anti-LFA1 alpha chain IT may remove accessory cells or stem cells critical to engraftment. Still, anti-LFA1-RTA may be useful for clinical GVHD prevention when combined with positive selection techniques designed to enrich for stem cells.

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