Page 1952 - Hematology_ Basic Principles and Practice ( PDFDrive )

1732 Part XI Transfusion Medicine

allogeneic HSCT can be used to enhance the GVN effect. 93,194 because it allows accurate interpretation of immunization results by
Appreciation of the GVN effect has led to the development of allowing a comparison between the detailed genetic makeup of the
nonmyeloablative stem cell transplants designed to immunosuppress individual receiving the vaccine and his or her antigen-specific
the host to a level sufficient to permit engraftment of the donor immune response. It is likely that in the future HLA laboratories will
immune cells to generate GVN without inducing the serious com- receive increasing demands for high-resolution, definitive typing for
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plications associated with myeloablation. The GVN effect has appropriate patient enrollment and for subsequent interpretation of
gained popularity in the last decade to the point that allogeneic-based immune responses.
immunotherapeutic approaches have been advocated for several
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nonhematologic malignancies. The rationale is largely based on the
assumption that the immune cell repertoire capable of recognizing MONITORING IMMUNE RESPONSES WITH TETRAMERIC
cancer cells in the allogeneic context is broader than in the autologous HUMAN LEUKOCYTE ANTIGEN-PEPTIDE COMPLEXES
system. Donor T cells can target not only tumor-specific antigens but
also allelic variants of these antigens; mHag; and, in the case of A growing understanding of the molecular immunology of T-cell
HLA-mismatched transplants, HLA antigens disparate from the interactions with HLA/epitope complexes in the context of infectious
donor expressed by the tumor cells. 195–197 Although there are several disease, virally induced malignancies, and spontaneous tumors as well
theories about how the GVN effect occurs, it remains unclear why as interest in their treatment with T cell-directed vaccines has gener-
allo-T cells have a better chance of targeting tumor cells in an allo- ated more attention to the issue of using accurate methods to quantify
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geneic context as compared with the natural insurgence of antitumor ex vivo the extent of antigen-specific immune responses. The most
immunity described for several solid tumors. commonly used assays available for the enumeration of antigen-
specific T cells include tHLA; intracellular fluorescence-activated cell
HUMAN LEUKOCYTE ANTIGEN AND sorter staining for cytokine expression on cognate stimulation; detec-
tion of cytokine release by enzyme-linked immunospot assay; and
T CELL-DIRECTED IMMUNIZATION quantitative real-time PCR, recently reviewed by Keilholz et al.
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tHLAs are complexes of four HLA molecules combined with a spe-
The last decade has witnessed remarkable progress in the identifica- cific peptide and bound to a fluorochrome (Fig. 113.6). These
tion and mapping of T-cell epitopes for various infectious diseases complexes bind to complementary TCR and identify antigen-specific
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and cancer. In particular, progress was made in mapping HLA- T cells, measuring cellular responses against specific epitopes with
+
associated epitopes for HIV, CMV, and Epstein-Barr virus. 198–201 In sensitivity as low as 1 in 5000 CD8 T cells. To synthesize tHLA
addition, the molecular identification of tumor-associated antigens molecules, soluble HLA heavy chains containing a biotinylation site
has yielded a large number of epitopes that could be used to immunize and recombinant β 2 -microglobulin are synthesized and purified.
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against neoplasia. A comprehensive discussion of these topics is They are then refolded in the presence of the specific epitope, and
beyond the scope of this chapter, but we address a few points framing the monomer is isolated by gel filtration and is biotinylated. Fluores-
the relevance of HLA in the context of T cell-directed immunization cent streptavidin is added to induce tetramerization. An aliquot of
(viral specific T cells). tetramers is added to the peripheral blood mononuclear cells together
The identification of T-cell epitopes led to two major areas of with other antibodies for a more detailed characterization of antigen-
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clinical investigation. The first is active-specific immunization to specific T cells. Analysis is performed using a flow cytometer.
prevent or treat ongoing infections or cancer. The second is the Because of the specificity of vaccines, the patient’s HLA type and
harvest and in vitro expansion of immunization-induced T cells for specific peptide must be identified and synthesized to provide the
adoptive transfer. In general, active immunization has proved suc- adequate tetramer. Pentameric HLA/peptide complexes have recently
cessful in inducing epitope-specific T cells easily detectable among become available and in some laboratories have replaced the use
circulating lymphocytes. 120,203–205 However, often the immunization- of tHLA.
induced enhancement of T-cell function is not associated with Analysis of tHLA offers many potential advantages over other
clinical improvement. Although the reason for the clinical inef- T-cell assays. This method is quantitative and enables an estimation
fectiveness of immunization-induced T cells is unclear, it has been of the avidity between TCR and peptide-loaded HLA class I mol-
postulated that they may be quantitatively or qualitatively inadequate ecules. tHLA staining does not kill the labeled cells, allowing sorting
for eradicating disease. 203,206 Therefore a second strategy is being of subpopulations by flow cytometry for additional analysis or expan-
pursued whereby the number of antigen-specific T cells is ampli- sion for adoptive transfer. With tHLA, specific T cells can be analyzed
fied in vitro for autologous or donor-derived adoptive transfer. This from blood samples without the prerequisite of in vitro culture, and
second strategy has met some promising success in the context of all specific cytotoxic T lymphocytes are detected, regardless of their
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ganciclovir-resistant CMV infection, Epstein-Barr virus–induced functional status. 203,206,213
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posttransplantation lymphoproliferative disorders, and metastatic
melanoma. 209
Whether delivered as a primary form of therapy or to prime in HUMAN LEUKOCYTE ANTIGEN SUMMARY
vivo T cells for further ex vivo expansion, epitope-specific vaccination
encounters the major limitation of a stringent requirement for HLA The relevance of HLA in clinical pathology has broadened from
allelic association. Although super families of HLA alleles may share its role as a predictor of allosensitization to a mediator of GVHD
epitopes, 14,16 in practical terms, clinically relevant HLA-epitope and GVN. The understanding of action mechanisms in various
associations are restricted to few peptide-allele combinations for a nonclassic HLA genes as well as KIR has opened a new field. The
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given protein. Patients considered for enrollment in immunization study examining the function of the innate immune response in the
protocols are best served by high-resolution HLA typing to exclude context of transplantation and other immunopathologies is a result
subtypes with unproven immunogenic potential for a given epitope. of this. Together with HLA, immunogenetics is rapidly growing,
To bypass the stringent HLA requirements demanded by epitope- driven by the realization that polymorphism is the hallmark of
specific vaccination, whole-antigen delivery is suggested. This is based human immunopathology as its spans mHag, KIR, cytokines, and
on the assumption that the epitope repertoire of a protein can be their receptors. Each of these is interwoven in an intricate array
adjusted to distinct HLA phenotypes by a cellular process of self- of interdependent functions that cannot be discounted. Modern
selection naturally coupling peptides to HLA according to binding methods should be able to adopt high-throughput systems for the
affinity. Although theoretically indisputable, in practice, the truth of parallel assessment of all these variables when addressing the genetic
this assumption depends on the efficiency with which individual makeup of an individual in correlation with the natural or therapeutic
molecules are processed and presented in association with distinct history of his or her disease. The comparison of donor and recipient
HLA alleles. Even in these settings, high-resolution typing is desirable protein profiling and cytokine polymorphism may offer insights