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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3011-3019
PLENARY PAPER
From the Department of Oncology, Johns Hopkins University School of
Medicine, Baltimore, MD.
For many cancers, autologous bone marrow transplantation (BMT)
achieves a minimal residual disease state, yet relapse rates remain high. Using a syngeneic murine bone marrow transplant
model, we demonstrate that vaccination with irradiated
granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing
autologous tumor cells is effective in the post-BMT period and actually
results in a greater tumor-free survival than vaccination in the
nontransplant setting. Employing T cells specific for a model
tumor-antigen, we find that transplantation of the tumor-bearing host
results in a massive expansion and activation of tumor-specific T cells in the early posttransplant period, but this response rapidly declines in association with tumor progression. Immunization with irradiated GM-CSF tumor cells during the period of immune
reconstitution results in the sustained amplification and
activation of this response that closely correlates with freedom
from relapse. These results demonstrate the feasibility of
integrating GM-CSF vaccines in the postautologous BMT setting and
suggest mechanisms that may contribute to the observed
efficacy of immunization during the critical period of immune reconstitution.
(Blood. 2000;95:3011-3019)
The past several decades have seen considerable
advances in the treatment of hematologic malignancies. These are in
large part attributable to the significant impact conferred by bone marrow transplantation (BMT). Dose escalation of cytotoxic therapy, together with improvements in supportive care, has resulted in prolonged disease-free survival and reduced transplant-related mortality. Despite this progress, a significant proportion of patients
transplanted for the treatment of hematologic malignancies will
eventually relapse and die of their disease.
A growing body of evidence suggests that immune-mediated effects may
contribute to tumor cell killing in patients with leukemia, lymphoma,
and multiple myeloma. In fact, the reduced relapse rates observed in
the setting of allogeneic transplantation when compared with autologous
BMT is likely the result of immune-mediated "graft-versus-tumor" effects. Unfortunately, because of the lack of tumor specificity of the
allo-response, what is gained by a reduction in relapse rates is lost
in the morbidity and mortality of graft-versus-host disease (GVHD) and
its treatment. Indeed, efforts to reduce the incidence and severity of
GVHD, such as more rigorous T-cell depletion of the grafts or more
intensive systemic immunosuppression of the recipient, are likely to
also reduce the magnitude of the allogeneic graft-versus-tumor effect.
Furthermore, the lack of a suitable donor or the advanced age of the
patient often precludes the option of allogeneic translantation. In
contrast, autologous transplantation has a relatively favorable
safety profile, while preserving the benefit of chemoradiation
used at doses beyond what can be given without stem cell support.
Accordingly, efforts to augment host antitumor immunity in the setting
of autologous transplantation may provide a means to diminish relapse
rates without a concomittant increase in toxicity.
Priming of systemic, tumor-specific immunity with vaccines against
tumor-associated antigens holds significant promise as a therapeutic
strategy. Animal models have demonstrated that immune responses can be
generated capable of eradicating small preestablished tumor burdens,
and early-phase clinical trials currently examining the
clinical efficacy of therapeutic cancer vaccines have reported the
induction of immune responses that are qualitatively similar to that observed in mouse models.1-5 Despite the enthusiasm
for this approach, substantial evidence suggests that the effect of active immunotherapy may be limited in the presence of advanced disease.6-8 Therefore, immunization in the adjuvant setting
may have the greatest potential to impact on tumor progression. For many malignancies, especially those of hematologic origin,
myeloablative chemotherapy and autologous BMT may offer the best chance
of achieving a state of minimal residual disease. Unfortunately, the
period of immune reconstitution following BMT has been characterized as
a time-decreased responsiveness to antigenic stimulation.9 Nevertheless, preclinical models10 and some clinical
trials11-14 have demonstrated the capacity of the
transplant recipient to respond to several different vaccine
formulations, underscoring the potential to manipulate host
antitumor immunity early during immune reconstitution. Indeed, the
normal homeostatic mechanisms that regulate adaptive immunity
are profoundly altered in the early posttransplant period, potentially
leading to augmented clonal expansion of antigen-specific lymphocytes
upon priming, "skewing" of thereconstituting T-cell repertoire
toward recognition of tumor-specific antigens,15 and
enhanced response to vaccination through the disruption of host
tolerogenic mechanisms.
We have used a mouse B-cell lymphoma model to develop strategies that
seek to integrate granulocyte-macrophage colony-stimulating factor
(GM-CSF)-based tumor cell vaccines in the postautologous BMT setting.
Immunization with irradiated autologous tumor cells engineered to
secrete GM-CSF has been shown to induce specific and long-lasting
antitumor immunity even against poorly immunogenic tumor models when
administered as a therapeutic vaccine in the treatment of small
established tumor burdens.16 We find that vaccination with irradiated GM-CSF-producing autologous tumor cells is
effective in the post-BMT period and actually results in a greater
degree of tumor-free survival than is achieved following vaccination
in the nontransplant setting. Mature T cells accompanying the graft
participate substantially in this response. In a minimal residual
disease model, tumor-specific T cells undergo a massive clonal expansion and activation in the early posttransplant period, which precipitously declines in close temporal association with the
development of macroscopic relapse. Vaccination with irradiated GM-CSF-producing tumor cells during immune reconstitution
substantially decreased the incidence of tumor relapse and was
accompanied by the persistence of an expanded population of activated
tumor-specific T cells. These studies suggest that a
"graft-versus-tumor effect" also occurs in the autologous BMT
setting, but it is not sustained. Repeated immunizations during immune
reconstitution may serve to maintain the increased precursor frequency
and activation state of tumor-specific T cells that is required to
prevent relapse.
Mice
Tumors cells
Syngeneic bone marrow transplantation The femurs and tibiae were obtained from 6- to 8-week-old donor BALB/c mice, and BM was harvested by flushing the bones with RPMI at 4°C. The marrow was treated with Low-Tox-M rabbit complement (Cedarlane Laboratories) for 30 minutes at 37°C in the presence of monoclonal antibody (MAb) J1J (anti-Thy1), MAb C3PO (anti-CD2), MAb RL172 (anti-CD4), and MAb 3-155 (anti-CD8) to obtain a T-cell-depleted BM. Single-cell suspensions were obtained from the spleens following FicollHypaque centrifugation. The graft consisted of 4 × 106 BM cells with or without the addition of 4 × 107 splenocytes (as indicated). Recipients were 6- to 8-week-old BALB/c mice that were irradiated with 850 cGy, followed by intravenous injection of the graft in a volume of 0.2 mL. The transplanted animals were maintained in sterile micro-isolator cages and received sterile food and water. Overall transplant-related mortality was less than 5%.Tumor purging of donor bone marrow and spleen In the indicated experiments, donor BM and splenocytes were depleted of A20 tumor by incubating with either an anti-IgG2a-biotin (Pharmingen, San Diego, CA) only or IgG2a-biotin, RA3.3A1-biotin (anti-B cell surface glycoprotein, B220), and 14.4.4-biotin (anti-I-Ed) for 30 minutes at 4°C in 2% fetal calf serum in phosphate-buffered saline. The cells were then incubated with streptavidin-conjugated Dynabeads (Dynal, Oslo, Norway) at a ratio of 4 beads/cell for 1 hour at 4°C and depleted by magnetic separation.Adoptive transfer Single-cell suspensions were made from peripheral lymph nodes and spleen that were harvested from TCR transgenic donors. The percentage of CD4+, TCR clonotype-positive lymphocytes was determined by flow cytometry as described below. Nontransplanted mice received 2.5 × 106 CD4+ anti-HA TCR T cells. In the BMT animals, the clonotypic T cells composed 1% of the splenic component of the graft.Vaccine preparation and administration A20/GM-CSF and A20HA/GM-CSF were created by retroviral transduction using the retroviral construct MFG-mGM-CSF as previously described.16 The cells were washed 3 times in sterile Hank's balanced salt solution, irradiated with 5000 cGy, and injected subcutaneously in the right flank (1 × 106 cells/0.1 mL).Tumor survival experiments All live tumor challenge with A20 wild-type or A20HA cells occurred via tail vein injection. In this model of systemic lymphoma, tumor traffics to spleen, mesenteric lymph nodes, liver, and can be found in blood and marrow at late stages. Progressive tumor is detected by the presence of increased abdominal girth and palpable splenomegaly, which is confirmed at autopsy by direct visualization. Ten mice were included per group, including unvaccinated controls, and each survival experiment was repeated at least once. Statistical analysis was performed using Kaplan-Meier survival and the log-rank (Mantel-Cox) test. Starview 4.5 software (San Francisco, CA) was used for the analysis.Flow cytometric analysis T cells were stained with fluorescein isothiocyanate-conjugated goat antimouse CD4 (Caltag, Burlingame, CA) and biotinylated rat anticlonotypic TCR MAb 6.5, followed by phycoerythrin-conjugated streptavidin (Caltag). A total of 50 000 gated events were collected on a FACScan (Becton Dickinson, San Jose, CA). Data represent the mean + SEM of the percentage of cells expressing the clonotypic TCR. Background staining of splenocytes from naïve BALB/c mice was less than 0.10%.
 -interferon (IFN)
production was measured by enzyme-linked immunosorbent assay (ELISA; R
& D Systems, Minneapolis, MN). Values for T cells cultured in media
alone were less than 10% of the values for HA peptide-stimulated T cells.
Kinetics of immune reconstitution and responsiveness to posttransplant immunization with irradiated GM-CSF-producing tumor cells The immediate posttransplant period is accompanied by significant immunosuppression likely resulting from the profound reduction in the number of normal immune effector cells. We sought to determine the point in time after BMT that a therapeutic vaccination could elicit an effective antitumor response. To directly compare the response to vaccination in the transplant versus nontransplant setting, we first established a model in which tumor was not present until after the recipient was irradiated, so that any differences observed between transplanted and nontransplanted mice would reflect the capacity of the immune system to respond to an equivalent tumor challenge. Transplants were staggered at weekly intervals (10 mice per group), followed by the intravenous challenge of all mice with 1 × 105 BALB/c-derived lymphoma cells (A20 wild type). Five days after tumor challenge, mice were vaccinated subcutaneously with 1 × 106 irradiated autologous tumor cells transduced to express GM-CSF (A20/GM-CSF). Normal BALB/c mice not having undergone a BMT were also challenged with this same tumor dose and vaccinated in an identical fashion (Figure 1A). Similar to our previously reported experience,18 vaccination of the nontransplanted cohort 5 days after tumor challenge conferred a 40% survival advantage over the nonvaccinated group (P < .04). In the BMT setting, early tumor challenge and vaccination (weeks 1 and 2) did not result in significant tumor rejection. However, a substantial antitumor effect of vaccination was observed at the 3-week point (70% long-term tumor-free survival). Indeed, the survival of mice challenged and immunized 3 to 6 weeks after BMT (40 mice in total) actually exceeded the survival of the nontransplant group given the identical tumor challenge and vaccination scheme (P < .03). A parallel kinetic analysis of lymphoid recovery revealed that the absolute number of CD4+ T cells (from pooled peripheral lymph nodes) 3 weeks post-BMT was less than 20% of that present in untransplanted ("normal") BALB/c mice, and CD8+ T cells were 33% of normal. By 6 weeks post-BMT, CD4+ T cells were 66% of normal and CD8+ T cells were 50% of normal. These results therefore demonstrate that a substantial response to this form of immunization can be generated prior to full immune reconstitution.
The enhanced antitumor effect in BMT is T-cell dependent While multiple cellular effector mechanisms participate in the response to vaccination with irradiated GM-CSF-producing tumor cells,16,20,21 a functional T-cell reservoir is absolutely required for the successful induction of tumor rejection.22 The early posttransplant period is characterized by profound alterations in the nascent immune repertoire, including that of B cells, NK cells, as well as T cells.9 To examine the contribution of T cells to tumor rejection in the BMT setting, transplants were carried out in either euthymic BALB/c mice or syngeneic athymic nude mice. In these experiments, the grafts consisted of T-cell-depleted BM alone, without the addition of mature T cells. Three weeks following BMT, these mice as well as unirradiated (nontransplanted) mice were challenged with A20WT tumor (1 × 106 intravenously) and followed for the kinetics of tumor progression (Figure 2). Tumor progression was most rapid in nude mice, irrespective of their transplant status. Given that NK cell function has been shown to be normal or even exaggerated in nude mice, these results suggest that enhanced NK activity during the posttransplant period cannot completely account for the improved tumor rejection previously observed. Tumor progression in euthymic mice transplanted with T-cell-depleted marrow was delayed relative to nude mice (with or without T-cell-depleted BMT, P < .001), suggesting that recent thymic emigrants may mediate some degree of antitumor effect in this experiment (where tumor challenge occurred 21 days post-BMT). Nevertheless, in contrast to transplantation with marrow plus splenocytes (Figure 1), the kinetics of tumor growth in euthymic mice transplanted with T-cell-depleted marrow alone was virtually identical to that observed in untransplanted BALB/c mice. These results provide evidence for T-cell-mediated antitumor immunity during the early phases of immune reconstitution, although tumor ultimately progresses in the absence of immunization.
Enhanced response of tumor-antigen-specific T cells to vaccination during immune reconstitution The above findings point to the repopulating T-cell compartment as a critical component of the antitumor response during the early posttransplant period. Given the favorable effects of vaccination with irradiated GM-CSF-transduced tumor cells during immune reconstitution, we wished to quantify the response of a defined tumor-specific T-cell population to vaccination in the transplant versus nontransplant settings. We used a well-characterized system employing the adoptive transfer of TCR transgenic CD4+ T cells specific for an MHC class II (I-Ed) restricted epitope of influenza HA. BALB/c mice underwent a syngeneic transplant consisting of T-cell-depleted BM plus splenocytes containing 1% HA-specific clonotypic T cells. Also present were nontransplanted (unirradiated) animals into which clonotypic T cells were adoptively transferred, as well as animals that underwent BMT in the absence of transgenic T cells. We examined the response of HA-specific T cells to immunization with a GM-CSF-producing tumor vaccine transfected to coexpress the model antigen (A20HA/GM-CSF). As a specificity control, a cohort of mice was vaccinated with A20/GM-CSF, which does not express HA. All animals were vaccinated 1 day following transplant (or adoptive transfer) and analyzed 14 days later. As we have previously observed in the nontransplant setting, priming with an irradiated GM-CSF-producing tumor vaccine expressing this model antigen fails to elicit a demonstrable clonal expansion of HA-specific T cells (Figure 3). In contrast, immunization with the identical vaccine in the posttransplant period resulted in a significant clonal expansion of antigen-specific T cells that accompanied the graft. Although T-cell repopulation of the peripheral compartment ultimately "dilutes" this percentage of clonotype-positive T cells, an expanded population of memory T cells was detectable 6 weeks after immunization (data not shown). These findings indicate that, even in the immediate posttransplant period, the host is capable of mounting a response to vaccination with irradiated GM-CSF-producing tumor cells as reflected in the antigen-specific clonal expansion of T cells that accompany the graft. Furthermore, radiation-induced changes in the recipient early after BMT permit a greater "burst" of T-cell expansion in response to immunization than occurs in immunized, nonirradiated mice.
Grafts from tumor-bearing donors can mediate a graft-versus-tumor effect The above results demonstrate that mature T cells accompanying the graft can participate in an endogenous graft-versus-tumor effect and are responsive to posttransplant vaccination. However, these experiments using tumor-free syngeneic donors may not accurately reflect alterations in T-cell function that could exist in grafts obtained from a tumor-bearing host. To more closely model the autologous transplant setting, we examined the effect of established tumor in the syngeneic donors. BALB/c mice to be used as donors were given 1 × 106 A20 cells 14 days prior to graft harvest or were left tumor-free. On the day of transplantation, donor marrow and splenocytes were harvested and "purged" of lymphoma cells by magnetic separation. Transplant recipients were challenged with 1 × 106 A20 cells 10 days prior to BMT, which was performed using grafts obtained from the tumor-bearing or non-tumor-bearing donors. This tumor burden, established prior to transplantation, utimately results in tumor progression in most mice reconstituted with grafts from naïve donors (Figure 4A). Surprisingly, however, grafts obtained from donor mice harboring A20 cells for 14 days prior to harvest actually mediated a substantial antitumor effect upon reconstitution of tumor-bearing recipients (P < .001). Furthermore, this unexpected result occurred in the face of incomplete elimination of tumor from the donor grafts, as demonstrated by the outgrowth of "contaminating" A20 cells upon in vitro culture of an aliquot of the graft after purging. Despite this, transplantation of most tumor-bearing recipients (as well as 5 of 5 non-tumor-bearing recipients data not shown) did not result in tumor growth in vivo, suggesting that failure to completely eliminate tumor from the graft
does not preclude long-term tumor-free survival of the recipient. These
results suggest that some fraction of the donor lymphocytes from
tumor-bearing mice are primed in response to tumor-antigen and remain
responsive upon transplantation. While it is possible that the
tumor-bearing donors in this experiment were not fully tolerant to A20
antigens, the tumor burden present when they were harvested
(1 × 106 A20 cells given 14 days earlier)
significantly exceeds what can be cured by vaccination alone in the
nontransplant setting (Figure 1B).
Endogenous activation of tumor-specific T cells during immune reconstitution Given the evidence for a T-cell-mediated graft-versus-tumor effect, we sought to examine the fate of tumor-antigen-specific T cells in the tumor-bearing transplant recipient during immune reconstitution. We employed a model in which tumor was established prior to transplantation to examine the consequences of radiation-induced tumor cell killing (and antigen release) as well as the effect of residual tumor present throughout the period of immune reconstitution. BALB/c mice were challenged with 1 × 106 A20HA cells 10 days prior to transplant or were left tumor-free. Following irradiation, they received grafts containing HA-specific TCR transgenic T cells. For comparison, nontransplanted (unirradiated) BALB/c mice with or without the same tumor challenge received an equivalent number of mature TCR transgenic T cells. Three weeks after BMT (or T cell transfer), mice were killed, and the percentage of HA-specific, clonotype-positive T cells was analyzed by fluorescence-activated cell sorter (FACS) (Figure 5). As we have previously reported, in the absence of BMT there was a modest increase in the percentage (from 0.45% to 0.78%) of clonotype-positive T cells upon adoptive transfer into tumor-bearing mice (Figure 5A). Despite this, these cells have been shown to have a markedly diminished capacity to proliferate and produce interleukin-2 and-IFN in response to the nominal peptide antigen in
vitro.23 In contrast to the nontransplanted tumor-bearing
mice, there was a dramatic clonal expansion of HA-specific CD4+ T cells in transplanted mice harboring A20HA (from
0.57% to 13.78%). The magnitude of this expansion far exceeded what
we have previously observed with any antigen-specific vaccine strategy
in the nontransplant setting and is substantially greater than the
response of non-tumor-bearing transplanted mice to vaccination with
irradiated A20HA/GM-CSF cells alone (Figure 3). Notably, at the time of
this analysis (day 21 post-BMT), there was no macroscopic evidence of
lymphoma present in the transplanted mice.
Immunization with irradiated GM-CSF-producing tumor cells during immune reconstitution sustains the activation of tumor-specific T cells The clonal expansion of tumor-specific T cells illustrated in Figure 5 occurred in a setting where parallel survival experiments demonstrated that tumor would ultimately progress in all unvaccinated mice. We therefore wished to examine the fate of these cells at a later time point as well as to determine the impact of posttransplant immunization on the state of tumor-specific T-cell activation. The experimental design was as described for Figure 5, with the addition of treatment groups that were immunized with irradiated A20HA/GM-CSF early (day +1 alone vs days +1, +8, +15, analysis day +21) or late (day +21 alone vs days +21, +28, +35, analysis on day +42). As before, 3 weeks after transplanting A20HA-bearing recipients there was a vigorous exansion of HA-specific CD4+ T cells (Figure 6A). The magnitude of this expansion was far greater than the effect of immunizing non-tumor-bearing transplant recipients with irradiated A20HA/GM-CSF. In fact, vaccination of tumor-bearing transplant recipients at the early time points did not result in a measurable increase in HA-specific T cell expansion beyond what occurred with transplantation alone. This clonal expansion was accompanied by substantial-IFN production in response to HA peptide in vitro,
indicative of differentiation into T-helper 1 (Th-1) effector cells
(Figure 6B). As before, 21 days after BMT no evidence of macroscopic
lymphoma was evident at dissection.
The impact of the graft-versus-leukemia (GVL) effect in allogeneic
stem cell transplantation25-28 and the positive results obtained with donor leukocyte infusions29-31 have led to an
increased recognition of the role played by the immune response in the
treatment of hematologic malignancies. Indeed, the capacity for
immune-mediated tumor cell killing of chemo-resistant cell
lines32,33 underscores the potential for this
non-cross-resistant treatment modality as an adjunct to dose-intensive
chemoradiation. Whereas there is much evidence to support the existence
of a T-cell-mediated graft-versus-tumor effect in the allogeneic
transplant setting, this effect has not been thought to contribute
significantly to tumor-free survival in autotransplants, which have
been largely viewed as a means to bypass the dose-limiting toxicities
of chemotherapy and radiation through "stem cell rescue."
Nevertheless, previous studies in mouse models have demonstrated that
the addition of syngeneic donor T cells to marrow grafts can
prolong tumor-free survival in a dose-dependent manner.19
Despite this effect, however, tumor relapse remains the major source of
failure for autologous BMT, and the immunologic events that accompany
tumor progression have been largely unexplored in this setting.
Submitted August 25, 1999; accepted January 3, 2000.
Supported by PHS grants CA15396-23 and CA78658-01.
I.B. is a Fellow of the Leukemia Society of America. E.M.S. is
a Fellow of the Lymphoma Research Foundation of America. H.I.L. is a
Scholar of the Leukemia Society of America.
Reprints: Hyam Levitsky, Johns Hopkins Oncology Center, 452 Bunting Blaustine Cancer Research Building, 1650 Orleans St, Baltimore,
MD 21231; e-mail: hy{at}jhmi.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
TCR specific for
influenza hemagglutinin (HA) peptide (amino acids 110-120) presented by
I-Ed were a generous gift of Harald von
Boehmer.
