Srinivas Viswanathan, Yi-Bin Chen
Hematopoietic stem cell transplantation (HSCT) has wide applications in the treatment of hematologic malignancies, congenital and acquired disorders of hematopoiesis, and autoimmune disease. Transplanted hematopoietic progenitors can reconstitute the full spectrum of hematopoietic cells in a recipient host, and can also confer immunologic antitumor activity. HSCT is therefore employed for both cellular replacement and for cancer immunotherapy. This chapter reviews the basic immunology of HSCT.
MAJOR CELLULAR TYPES
Following HSCT, donor hematopoietic progenitor cells migrate to the recipient bone marrow and differentiate to generate all cell types of the erythroid, myeloid, and lymphoid lineages. The following hematopoietic cell types are thought to play key physiologic roles:
• CD34+ cells: The population of cells containing the hematopoietic progenitors capable of repopulating the recipient marrow. HSCT grafts are usually quantified based on the CD34+ population. In certain selective cellular depletion protocols, donor hematopoietic cells can be purified for CD34+ cells prior to transplantation. Doses of more than or equal to 2 × 106/kg (recipient weight) CD34-expressing progenitor cells are typically required in autologous or adult allogeneic adult donor products. Requirements are approximately one log lower for umbilical cord products.
• B-cells: Lymphocytes whose chief role is to produce antibodies against specific antigens to mediate humoral immunity. The role of B cells in chronic graft-versus-host disease (cGVHD) has been recognized.
• CD8+ T cells: Cytotoxic T cells that recognize antigenic peptides presented on major histocompatibility (MHC) Class I proteins. In the presence of either a costimulatory signal or stimulatory cytokines (secreted by CD4+ Th and other cells), CD8+ T cells undergo clonal expansion and lyse their targets via the release of perforin and granzyme.
• CD4+ helper T (Th) cells: Helper T cells that can develop into either Th1 or Th2 effector T cells. Th1 cells support the cellular immune response by stimulating killing by macrophages and CD8+ T cells. Th2 cells support the antibody response by stimulating B-cell proliferation and antibody production. A third subset of Th cells (Th17 cells) has been identified although its role in HSCT is yet to be defined.
• Natural killer (NK) cells: Cytotoxic lymphocytes central to the innate immune response. NK cells kill cells that have lost cell-surface expression of self MHC I in a perforin- and granzyme-dependent manner. NK cells are also key effectors in humoral immunity, mediating antibody-dependent cellular cytotoxicity (ADCC).
• Regulatory (Treg) cells: A subset of CD4+ T cells that functions in a regulatory capacity to modulate the immune response and suppress autoimmunity. Ongoing clinical trials are exploring different ways to expand Treg cells to control GVHD.
• Dendritic cells: Antigen-presenting cells (APCs) that express high levels of both MHC I and MHC II and efficiently present antigens to T cells. Dendritic cells stimulate naïve T cells within lymphoid tissues. Dendritic cells within the thymus eliminate T cells that are selective for self-antigens through the process of negative selection.
GRAFT-VERSUS-HOST DISEASE
Graft-versus-host disease (GVHD) results from an immunologic response against antigenic disparities between donor hematopoietic cells and host tissues. GVHD comprises a significant proportion of morbidity and mortality associated with HSCT. Historically, GVHD had been classified into acute (traditionally occurring within 100 days of transplant) and chronic (traditionally occurring after 100 days of transplant) phases. However, recent consensus definitions have transitioned to a classification based solely on clinical manifestations, rather than the timing of symptoms after HSCT. There is also an acute-chronic GVHD overlap syndrome which has manifestations of both classic acute and chronic disease (Table 37-1).
TABLE 37-1 COMPARISON OF FEATURES OF ACUTE GVHD AND CHRONIC GVHD
Significant acute GVHD (aGVHD) complicates up to half of all HLA-matched stem cell transplants and a higher proportion of mismatched transplants. The most commonly involved sites of aGVHD are the skin, liver, and gastrointestinal tract. Clinical features include a maculopapular rash, liver function test abnormalities (traditionally, elevations in direct bilirubin and alkaline phosphatase), and high-volume diarrhea accompanied by abdominal cramping. Histologic features differ by organ, and it is worth noting that the overall clinical picture, and not pathologic findings, is the gold standard for diagnosis. Skin involvement by aGVHD is characterized histologically by dermal and epidermal lymphocytic infiltration. Liver aGVHD is characterized by lymphocytic infiltration of small bile ducts, leading to bile duct damage and degeneration, and intestinal aGVHD is characterized by crypt cell necrosis, increased apoptosis, and a loss of intestinal epithelium (1, 2).
The pathogenesis of aGVHD is thought to be driven by donor T-cell-mediated damage of host cells as well as by local and systemic release of inflammatory cytokines. This is thought to occur via a multistep process: (1) Damage to host tissues by the conditioning regimen (classically, high doses of chemotherapy and radiation) stimulates the release of proinflammatory cytokines including IL-1, IL-6, TNF-α, and IFN-γ (2) increased MHC molecule expression on host APCs including dendritic cells in response to cytokines; (3) presentation of alloantigens to donor T cells by activated host APCs triggers T-cell activation, leading to IL-2 and IFN-γ production; (4) CD4+ T cells stimulate expansion of the donor CD8+ T cells that mediate the cytotoxic effects of GVHD; and (5) direct damage by the cytotoxic actions of activated donor CD8+ T-cells and inflammatory cytokines result in the clinical manifestations of acute GVHD (Figure 37-1).
Figure 37-1 Schematic of the steps leading to GVHD. In the afferent phase, damage to host tissues by conditioning regimen leads to inflammatory cytokine production and upregulation of MHC on host APCs. Subsequently, recipient and donor APCs activate donor-derived T cells. Cytotoxic (CD8) T cells then cause damage to host tissues.
Every allogeneic HSCT requires some form of prophylaxis against GVHD. This can be accomplished through (1) pharmacologic methods (most commonly) or (2) ex vivo T-cell depletion methods. The accepted international standard for pharmacologic prophylaxis against aGVHD involves a calcineurin inhibitor (either cyclosporine or tacrolimus) in combination with several low doses of post-HSCT methotrexate. Calcineurin inhibitors are generally continued at therapeutic levels for several months with gradual tapering to discontinuation if there are no signs of GVHD.
The initial treatment for patients who develop clinical aGVHD despite appropriate prophylaxis involves high-dose systemic glucocorticoids, usually at doses of 1–2 mg/kg/day of prednisone or its equivalent. Glucocorticoids provide durable remission in only 50% of patients, and those who fail initial therapy have high mortality rates (3, 4). There is no standard second-line therapy and a number of agents have been tried, including mycophenolate mofetil, sirolimus, IL-2 antagonists, TNF-α antagonists, pentostatin, and extracorporeal pheresis.
Chronic GVHD (cGVHD) develops in a subset of patients with acute GVHD, and also arises in the absence of any preceding acute GVHD. The most commonly involved sites are the eyes, skin, respiratory tract, esophagus, and liver. Clinically, chronic GVHD is pleomorphic and heterogeneous, with many features resembling classic autoimmune diseases such as lupus, Sjogren’s syndrome, vitiligo, and scleroderma. In contrast to the inflammatory changes that are typically found in aGVHD biopsies, pathologic hallmarks of cGVHD include a dense fibrosis of involved tissues with occasional infiltration of mononuclear cells (5).
Both donor B cells and donor T cells appear to play important roles in the pathophysiology of chronic GVHD. T-cells have been implicated in cGVHD in multiple mouse models, as well as by the fundamental observation that cGVHD incidence is significantly less in patients who undergo transplants from T-cell depleted grafts. The role of B cells has emerged recently from studies showing antibodies specific for proteins coded from the Y chromosome in female donor—male recipient transplants (6). In addition, other studies have also demonstrated that B-cell activating factor (BAFF), a pro-B-cell growth factor, is present at high levels in patients with active chronic GVHD (7).
Many of the same agents used for treatment of aGVHD are also used for the treatment of cGVHD, including systemic glucocorticoids, calcineurin inhibitors, mycophenolate mofetil, sirolimus, and extracorporeal pheresis. Given the recent data implicating the role of B cells in cGVHD, the anti-CD20 antibody rituximab has also been employed with encouraging results (8). Chronic GVHD has emerged as the most important determinant of quality of life in long-term survivors of HSCT (9), and, unfortunately, many trials that have shown some success in reducing acute GVHD have not had a significant effect on preventing chronic GVHD. Therefore, one of the primary focuses of future research in HSCT involves better prevention and treatment of chronic GVHD.
GRAFT-VERSUS-LEUKEMIA
The existence of an immunologic graft-versus-leukemia (GVL) effect has been supported by several observations: (1) with comparable conditioning regimens, allogeneic transplants result in a lower relapse rate than autologous or syngeneic transplants; (2) multiple series have observed that patients who develop acute or chronic GVHD have a lower incidence of disease relapse than those who do not (10); and (3) donor leukocyte infusions (DLI) alone were able to achieve remission for many patients with relapsed chronic myeloid leukemia (CML) after HSCT (11). Although antitumor effects of HSCT have been most widely studied in the setting of leukemia, regression of other hematologic malignancies has also been clearly described following HSCT, leading to the use of the broader term graft-versus-malignancy (GVM). The recognition of the GVM effect has led to the increasing popularity of protocols employing nonmyeloablative and reduced-intensity conditioning regimens. The efficacy of these protocols is based mostly on the antitumor effects of GVM, with toxicity spared due to the lower intensity of the conditioning regimens.
The extent or potency of GVM appears to vary based upon the underlying disease, disease status, and, undoubtedly, unknown essential interactions between the donor and the recipient. Both donor-derived T cells and NK cells appear to have key roles in GVM. In theory, the T-cell response is driven by the presentation of host or tumor antigens to donor T cells, which leads to a clonal expansion of CD8+ cytotoxic T cells specific for the antigen. NK cells also appear to be potent mediators of GVL (12, 13), but differ from T cells in that they can kill tumor cells without the prerequisites of activation and clonal expansion. NK cells express inhibitory receptors known as killer cell immunoglobulin-like receptors (KIRs) which recognize specific inhibitory MHC Class I allele groups (KIR ligands). NK-cell alloreactivity occurs when recipient cells do not express an MHC-I ligand which can engage KIR on the surface of donor NK cells. Overall, it remains unclear if the immunologic mechanisms driving GVM differ from those responsible for GVHD, and current methods are unable to reliably separate the two (14). Ongoing research is focusing on methods to better cultivate GVM without inducing significant GVHD, using approaches such as preemptive post-HSCT treatment with immunomodulatory agents and vaccination protocols against specific tumor antigens. A summary of these approaches is shown in Figure 37-2.
FIGURE 37-2 The promise of HSCT lies in augmenting GVL (thick arrow) while minimizing GVHD (dotted arrow). Strategies for minimizing GVHD include pharmacologic agents, T-cell depletion, increasing number of Treg cells, extracorporeal pheresis (ECP), and mesenchymal stem cell (MSC) infusion. Strategies for augmenting GVL include donor-lymphocyte infusion (DLI), increasing number of Treg cells, conducting NK-cell (KIR) mismatched transplants, tumor vaccines, and therapies targeted and tumor-specific antigens.
GVHD PROPHYLAXIS
PHARMACOLOGIC APPROACHES
As mentioned previously, the accepted standard pharmacologic approach to prevent GVHD includes a calcineurin inhibitor in combination with several doses of post-HSCT methotrexate. Other agents have been added to this backbone with the hope of preserving GVM, while preventing GVHD more effectively. Several of these agents are highlighted below.
Antithymocyte globulin (ATG) is very commonly given during conditioning to help prevent GVHD by depleting donor T cells in vivo. Several different ATG preparations exist, and they differ in their specificity and activity. Many US centers routinely employ thymoglobulin (ATG, Genzyme) when using unrelated or mismatched donors (15), although no prospective clinical trials have shown a proven clinical benefit (16). Recently, a large European study using ATG-F (Fresenius) in addition to cyclosporine and methotrexate showed a decreased incidence of both aGVHD and cGVHD without increasing the rate of relapse (17), and a large confirmatory American trial is now underway.
Alemtuzumab is a humanized monoclonal antibody against CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein expressed on the surface of B and T lymphocytes, NK cells, macrophages, and dendritic cells. In vitropre-treatment of donor cells with alemtuzumab prior to transplant as a form to T-cell depletion has been performed (18). More commonly, alemtuzumab is included in conditioning regimens to provide a form of in vivo T-cell depletion with all matched (19), mismatched (20), and haploidentical (21) donors. Results of these series all show impressively low rates of GVHD, yet there is a clearly higher incidence of infections and possibly relapse of disease.
Sirolimus (rapamycin) has recently emerged as a promising immunosuppressive agent which works through several mechanisms: (1) binding of FK-binding protein 12 (FKBP-12) leading to inhibition of the mTOR pathway, thus blocking activation of B cells and T cells; (2) blocking antigen presentation and dendritic cell maturation; and (3) promoting the development of regulatory T cells. A number of phase II studies suggest that sirolimus may be beneficial in the treatment of both aGVHD and cGVHD, as well as in GVHD prophylaxis (22), and a large national collaborative phase III trial in GVHD prophylaxis has recently finished accrual with results eagerly awaited.
Cyclophosphamide, an alkylating agent used commonly in the treatment of many malignancies, selectively induces apoptosis in proliferating T cells. High-doses (50 mg/kg/day on days +3 and +4 after HSCT) given in the first week after HSCT has been pioneered as a novel method to prevent GVHD and promote tolerance. This was first developed in murine models and has been successfully translated into human patients using both matched related (23) and haploidentical donors (24).
Newer, more experimental, approaches seek to manipulate the cytokine environment. Both antitumor immunity and aGVHD appear to be dominated by a Th1-polarized T-cell response (with key cytokines being IL-1, IL-2, IL-6, IFN-γ, TNF-α), while cGVHD is driven by a Th2-polarized response (with key cytokines being IL-4, IL-5, IL-10). Several attempts have been made, all in preclinical animal models, to alter the cytokine milieu in such a way as to minimize GVHD while preserving GVM. While these approaches are promising, it remains unclear to what extent exogeneous cytokine administration can be safely translated to the human clinical setting.
Anergy refers to the process whereby immune tolerance is induced. Several attempts have been made to induce anergy in donor T cells (“alloanergy”) and thereby reduce the incidence of GVHD. Most approaches to doing this focus on blocking the costimulatory signal between host APC and donor T cells by using monoclonal antibodies or fusion proteins (25). In vitro results employing this approach are promising, suggesting that alloreactive T cells are reduced in number without major effect on GVM. Clinical trials of this alloanergization approach are currently in progress.
CELL-BASED APPROACHES
Cellular approaches to preventing GVHD have largely centered on strategies attempting to selectively deplete the donor T cell pool of alloreactive T cells. Early strategies employed ex vivo pan-T cell depletion using antisera directed against all T cells. While this approach reduced the incidence of GVHD, relapse rates and infectious complications were increased, leading to comparable rates of overall survival relative to traditional pharmacologic methods. In appropriate clinical settings, ex vivo T-cell depletion remains a very effective method of GVHD prophylaxis (26, 27). However, rigorous T-cell depletion methodology requires significant laboratory expertise and facilities.
Preemptive donor lymphocyte infusion (DLI) has been used in an attempt to augment the GVM effect while minimizing GVHD. When DLI is used for this purpose, it is typically given to patients who have received in vivo or ex vivo T-cell depleted transplants, especially if those patients exhibit evidence of mixed donor-host chimerism posttransplant (28). DLI clearly has the capacity to convert mixed host-donor chimerism to full donor chimerism without causing graft rejection (28, 29). Because the acute inflammation that activates host antigen presentation subsides after the immediate posttransplant period, this technique theoretically reduces allogeneic T-cell reactivity while preserving GVM. In practice, however, the timing of DLI is often coincident with immunosuppression taper, which may also contribute to promoting GVHD.
DLI is also commonly employed as salvage immunotherapy following disease relapse after HSCT. Historically, when given to several patients with relapsed chronic phase CML after HSCT, DLI alone was able to induce remission—one of the earliest examples of a clear GVM effect. Nonetheless, more recent data have shown that DLI is generally ineffective if a significant burden of disease is present for the majority of diseases. Furthermore, there is a significant risk of developing GVHD after DLI (30, 31). Clinical trials are investigating techniques to increase the effectiveness of DLI while suppressing the risk of GVHD. These strategies include the insertion of “suicide” transgenes into donor lymphocytes prior to infusion to allow a simple and controlled means for killing alloreactive T cells after transplantation if significant GVHD develops (32, 33). Other approaches include giving selective populations of DLI and coadministration of specific cytokines to increase GVM activity.
There has been great interest in the regulatory T-cell subset as a means to separate GVL from GVHD. Tregs comprise about 5% of the CD4+ T-cell pool and express high levels of CD25 and FoxP3. In multiple mouse models, Treg cells can potently suppress aGVHD without impairing GVM. Therefore, augmenting the activity or size of the donor Treg cell pool is an attractive way to enhance GVM and minimize GVHD. However, reliable and generalizable methods of ex vivo expansion of Tregs are not currently available, limiting the ability to translate promising preclinical results to the clinical setting although clinical protocols are ongoing (34).
Mesenchymal stem cells (MSCs) are bone marrow derived stromal cells with the capacity to differentiate into fibroblasts, adipocytes, osteoblasts, and chondrocytes. In various preclinical models, MSCs have been shown to stimulate the production of regulatory T cells and suppress development of GVHD. This is likely mediated through the secretion of particular cytokines or growth factors that stimulate Treg cell growth and development. Early clinical data indicated that MSCs were safe for use in humans and may improve outcomes in steroid-resistant acute GVHD (34, 35), and although this has not yet been confirmed in larger prospective studies, use of MSCs remain an active area of investigation.
Extracorporeal photopheresis (ECP) is an immunomodulatory procedure used for the treatment of cGVHD and, more recently, aGVHD. With ECP, the patient undergoes leukapheresis and collected leukocytes are treated with a DNA-intercalating dye. The leukocytes are then exposed to UVA radiation, which causes cells to undergo apoptosis. Following dye treatment and UVA exposure, treated leukocytes are returned to the patient. Data from murine models indicate that ECP acts through several mechanisms: promoting alloreactive lymphocyte apoptosis, increasing numbers of regulatory T cells, and indirectly decreasing the number of donor effector lymphocytes that have never even been exposed to dye or UVA radiation (36). ECP is especially attractive in that it appears to induce less global immunosuppression relative to other therapies.
Umbilical cord blood (UCB) stem cells are the newest source of hematopoietic stem cells used in HSCT. UCB transplantation is attractive as less stringent HLA-matching requirements are required given the intrinsic immunologic immaturity of UCB cells. With the growth of UCB transplantation, several retrospective series have suggested that rates of acute and chronic GVHD are lower compared to adult peripheral blood stem cells or bone marrow, yet relapse rates are lower as well. However, overall rates of survival remain comparable given higher rates of mortality from infections and other complications with UCB transplantation. Nevertheless, it is compelling that rates of relapse are not increased in this setting where GVHD is clearly decreased.
CONCLUSIONS
Hematopoietic stem cell transplantation serves as a powerful technique for both cellular replacement and for cancer immunotherapy. Unfortunately, the immunologic anti-malignancy effects of HSCT are often offset by debilitating effects of GVHD. Work has begun to elucidate the myriad cell types and complex molecular pathways involved in both GVL and GVHD. It is only after understanding the specific immunologic players involved in each of these processes that we will be able to develop therapeutic approaches which can potentially separate them. Although recent approaches have suggested the ability to partially decrease GVHD without increasing infection or disease relapse, future work is clearly needed to further refine HSCT into an adoptive immunotherapy platform with more tolerable morbidity and mortality.
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