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Ann Thorac Surg 2003;75:S58-S65
© 2003 The Society of Thoracic Surgeons

Supplement

Interactions between the recipient immune system and the left ventricular assist device surface: immunological and clinical implications

Silviu Itescu, MDa*, Ranjit John, MDa

a College of Physicians and Surgeons of Columbia University, New York, New York, USA

* Address reprint requests to Dr Itescu, Transplantation Immunology, Department of Surgery, College of Physicians & Surgeons of Columbia University, 622 West 168th St, P&S 14-402, New York, NY 10032, USA.
e-mail: si5{at}columbia.edu

Presented at the Heart Failure & Circulatory Support Summit, Cleveland, OH, Aug 22–25, 2002.

Abstract

The unquestionable clinical success of left ventricular assist device (LVAD) implantation has, nevertheless, been accompanied by complications arising from interactions between the implanted biomaterial and the host immune system. The aberrant state of monocyte and T-cell activation resulting from these host/device interactions is accompanied by two parallel processes: (1) selective loss of Th1 cytokine producing CD4 T-cells through activation-induced cell death; and (2) unopposed activation of Th2 cytokine producing CD4 T-cells resulting in B-cell hyperreactivity and dysregulated immunoglobulin synthesis through Th2 cytokines and heightened CD40 ligand-CD40 interactions. The net results of these events is that, on the one hand, the LVAD recipient develops progressive defects in cellular immunity and is at increased risk of serious infection, and, on the other hand, is more likely to develop allosensitization, posing a significant risk to successful transplant outcome. Intravenous immunoglobulin therapy is an effective and safe modality for sensitized LVAD recipients awaiting cardiac transplantation, reducing serum anti–human lymphoicyte antigen (HLA) alloreactivity and shortening the duration to transplantation. The therapeutic and safety profile of intravenous immunoglobulin would appear to be superior to plasmapheresis. Immunosuppression incorporating intravenous cyclophosphamide before and after transplantation is safe and highly effective in sensitized LVAD recipients of cardiac transplantation. When used after transplantation as part of triple immunosuppressive regimens, cyclophosphamide is superior to mycophenolate mofetil in reducing episodes of allograft rejection in these patients. Because these immune dysfunctions appear to be related to the effects of excessive biomaterial-associated T-cell activation, future efforts will need to be directed at either altering the physical properties of the materials interacting with the host circulation or pharmacological intervention aimed more selectively at inhibiting T-cell activation.

The development of novel materials used for implant surgery and the increasing use of implanted devices has made it evident that no material is biologically inert and that optimal use of such biomaterials requires improved knowledge of events occurring at the host/implant interface. Biocompatibility may be considered in terms of four separate but interrelated components: the adsorption of proteins and other macromolecules on the material surface, changes in the material induced by the host, local effects of the material on host tissues, and systemic or remote effects of the material on the host. Commonly utilized biomaterials, including so-called inert compounds such as titanium, polytetrafluoroethylene (PTFE), and acrylics, may trigger an array of iatrogenic effects, including inflammation, fibrosis, coagulation, and infection. A localized host inflammatory response is a common occurrence irrespective of the material used, and such responses may impact adversely on the implant, for example, osteolytic changes around joint implants, stress cracking of pacemaker leads, and fibrosis surrounding mammary prostheses. In situations where the biomaterial is in direct contact with the blood circulation, such as with hemodialysis, significant changes in systemic immunologic and thrombostatic functions have been described.

The discrepancy between the limited availability of donor organs [1, 2] and the ever-increasing number of patients with heart failure has led to the development of left ventricular assist devices (LVADs). LVADs are being increasingly used as bridges to cardiac transplantation, with satisfactory survival rates [3–5]. The encouraging medium-term results with implantable LVAD support have stimulated the initiation of prospective, randomized, multicenter trials evaluating permanent LVAD implantation as a therapeutic modality for patients with end-stage heart failure. Because the biomaterials on the LVAD surface are exposed to the entire host circulation, it becomes mandatory to define the biology of the host-LVAD relationship beyond life-sustaining pump, and to determine the effects of LVAD implantation on systemic host immunity. In this review, we discuss the effects of the LVAD on the host immune system and the clinical consequences in LVAD patients. It is important to note that the results of the studies shown below were all performed in patients with the Heartmate LVAD system, unless specified otherwise.

Effects of the LVAD/host interface on localized elements of the host immune system

Deposition of monocytes/macrophages on LVAD surface
The clinical success of LVAD implantation has nevertheless been accompanied by significant complications, including thromboembolic events in as many as 30% of cases [6, 7]. This complication is reduced to less than 4% [8] in LVAD types that incorporate a textured surface lining that supports the growth of neointima-type cells [9, 10]. To begin to understand the nature of this cellular lining, we investigated the cellular phenotypes present on the LVAD surface. The majority of the cells detected after cell detachment and resuspension were of monocyte/macrophage lineage. Resting monocytes or activated macrophages were identified, respectively, as either round, quiescent CD14-positive cells or elongated, often multinucleated, CD68-positive cells with prominent cytoplasmic processes. The macrophage lineage cells were functionally activated as defined by NFkB expression [11] and augmented production of cytokines and coagulation factors [12].

Monocyte–T-Cell interactions on the LVAD surface result in aberrant T-Cell activation
Interspersed among the cells of monocyte/macrophage lineage were T lymphocytes, which expressed strong immunoreactivity for CD3, CD4, and CD25 (interleukin 2[IL-2] receptors), consistent with activated helper T-cells [13]. Moreover, incubation of T-cells from the LVAD surface with LVAD material and exogenous IL-2 caused a sevenfold increase in T-cell proliferation compared with culture in medium alone, consistent with expansion of in vivo activated T-cells. Similarly, T-cell aggregates from the LVAD surface could be sustained in culture for up to 3 weeks in the presence of IL-2 and LVAD material, but not in the absence of either. Finally, using the reverse-transcriptase polyacrimade chain reaction (RT-PCR) technique to determine the cytokine profile expressed by the neointimal cells on the LVAD surface, all samples studied demonstrated mRNA for both the Th1 cytokine IL-2 and the Th2 cytokine IL-10, as well as for the monocyte-derived cytokine IL-1 [13]. Together, these observations emphasize the monocyte–T-cell interactions that occur on the LVAD surface, and indicate that the consequences of these interactions are to induce prominent T-cell activation through IL2 receptor–dependent pathways.

Systemic consequences of aberrant immune activation accompanying LVAD implantation: induction of T-Cell defects and B-Cell hyperreactivity

Circulating T-cells from LVAD recipients demonstrate heightened levels of CD95 (Fas) expression, spontaneous proliferation, and spontaneous apoptosis in vivo
To determine whether this aberrant T-cell activation on the LVAD surface influenced systemic T-cell immunity in LVAD recipients, we next examined the phenotype and function of circulating T-cells in LVAD recipients. Circulating T-cells from LVAD recipients demonstrated a heightened state of in vivo activation, as defined by surface expression of the activation marker CD95 (Fas), associated with a pathway of cellular apoptosis. CD95 expression was increased to a similar extent on both CD4 and CD8 T-cells from LVAD recipients in comparison with controls, (70% ± 6% vs 22% ± 4%, p < 0.001; and 69% ± 7% vs 7% ± 2%, p < 0.001, respectively). Reflecting this heightened state of in vivo activation, T-cells from LVAD recipients had significantly higher levels of spontaneous proliferative activity in comparison with T-cells from New York Heart Association (NYHA) class IV controls, and this activity increased in parallel with duration of LVAD implantation.

We next investigated whether this heightened state of T-cell activation in LVAD recipients was associated with increased levels of T-cell apoptosis in vivo. Spontaneous T-cell apoptosis, defined by binding of annexin V to phosphatidylserine present on T-cell membranes of cells undergoing early phases of apoptosis, was significantly higher in both CD4 and CD8 T-cells from LVAD recipients than in those from NYHA class IV heart failure controls (39% ± 5% vs 4% ± 1%, p < 0.001; and 45% ± 4% vs 2% ± 1%, p < 0.001, respectively). Similar abnormalities were observed on T-cells from recipients of either TCI Heartmate or Novocor devices. This enhanced state of T-cell apoptosis in vivo was confirmed by analysis of DNA isolated from freshly obtained T-cells. A characteristic fragmentation pattern of apoptosis was observed in DNA from circulating T-cells of all LVAD recipients, but not in DNA from T-cells of any control individuals.

Circulating t-cells from LVAD recipients demonstrate defective proliferation after triggering via the T-Cell receptor complex
Despite the high levels of spontaneous proliferation, T-cells from LVAD recipients showed defective proliferative responses after activation, specifically, through the T-cell receptor (TCR) complex. After TCR engagement by allogeneic mixed lymphocyte culture, the mean stimulation index (SI) of T-cells from LVAD recipients was 74% lower than that of T-cells from NYHA class IV controls (p < 0.001). Similarly, after TCR ligation with anti-CD3 monoclonal antibody (Mab), the mean SI of T-cells from LVAD recipients was 83% lower than that of T-cells from NYHA class IV controls (p < 0.001). In contrast, T-cell activation by pathways other than TCR triggering caused similar increases in T-cell proliferation in both LVAD recipients and controls.

Circulating T-Cells from LVAD recipients demonstrate increased susceptibility to activation-induced cell death (AICD) after T-Cell receptor engagement
Because preactivated T-cells expressing CD95 (Fas) are susceptible to activation-induced cell death (AICD) after triggering through the TCR complex, we next investigated whether the observed defects in T-cell proliferative responses in LVAD patients after TCR engagement might be related to AICD. A flow cytometric assay was used to detect the proportion of apoptotic T-cells in a given individual (LVAD recipient or NYHA class IV control), defined by annexin V binding to surface phosphatidylserine, which underwent cell death, defined by propidium iodide staining, after 24 hours of culture with either medium or anti-CD3 Mab. The increase in T-cell death after anti-CD3 activation was then compared in both experimental groups: LVAD patients and NYHA class IV controls. After activation of resting T-cells with anti-CD3 Mab the proportion of annexin V–positive CD4 T-cells undergoing cell death (propidium iodide positive) increased by a mean of 3.2-fold among LVAD patients compared with only 1.2-fold in heart failure controls (p < 0.05). These results clearly demonstrate that circulating CD4 T-cells from LVAD recipients have increased susceptibility to AICD in comparison with those from heart failure controls.

Alterations in T-cell cytokine profiles in LVAD recipients: loss of Th1, but not Th2, cytokine mRNA expression
Because T-cells producing Th1 type cytokines (IL-2 and interferon [IFN]-gamma) have been reported to be selectively susceptible to CD95 (Fas)–mediated apoptosis [14, 15–18], we next used the RT-PCR technique to compare the pattern of Th cytokine gene expression in LVAD recipients and heart failure controls. Freshly obtained circulating PBMC from each of 12 NYHA class IV control patients expressed mRNA for both Th1 type cytokines (IL-2 and IFN-gamma) and Th2 type cytokines (IL-10 and tumor growth factor [TGF]-beta). In contrast, freshly obtained PBMC from each of 12 LVAD recipients expressed mRNA for Th2 type cytokines (IL-10 and TGF-beta), but not for the Th1 type cytokines IL-2 and IFN-gamma. Expression of IL-4 or IL-5 mRNA was not detected in any LVAD or control patient. These results suggest that the heightened levels of T-cell apoptosis in LVAD recipients leads to a selective loss of T-cells producing Th1-type cytokines and unopposed T-cell production of Th2-type cytokines.

B-cell hyperreactivity in LVAD recipients: high frequency of antiphospholipid and anti-HLA antibodies
Because induction of autoimmunity, polyclonal B-cell activation, and production of autoantibodies have been postulated to result from both excessive circulating apoptotic waste [19–21] and from a predominance of circulating Th2-type cytokines [22, 23], we investigated whether LVAD recipients demonstrate prominent B-cell hyperreactivity. LVAD recipients had significantly higher frequencies of circulating antiphospholipid and anti-HLA antibodies in comparison with NYHA class IV controls awaiting cardiac transplantation. Circulating antiphospholipid antibodies were detected in 9 of 20 LVAD recipients (45%), but in none of 20 heart failure controls (p < 0.0001). Similarly, the frequencies of IgG antibodies against major histocompatibility complex (MHC) class I and class II antigens were significantly higher in LVAD recipients than in heart failure controls awaiting transplantation (43% vs 3% and 33% vs 3%, respectively, both p < 0.0001).

Presence of HLA-DR3 predisposes LVAD recipients to B-cell hyperreactivity
We next sought to determine whether production of anti-MHC antibodies in LVAD recipients was influenced by either perioperative transfusion of blood products or by host genetic factors. Sixty-three percent of patients who received more than 6 platelet units were found to develop IgG antibodies against MHC class I antigens by 4 months of LVAD implantation compared with 8% of those receiving less than 6 U (p < 0.01). Perioperative red blood cell transfusions did not influence the production of these antibodies, presumably because donor red blood cells contain less contaminating MHC class I–expressing T-cells than donor platelets. In contrast to anti-MHC class I antibodies, development of IgG antibodies against MHC class II antigens was not influenced by either the number of perioperative platelet or red blood cell transfusions, presumably because contaminating T-cells in the absence of activation do not express MHC class II antigens.

We next investigated whether development of anti–MHC class II antibodies in LVAD recipients was influenced by inheritance of particular HLA-DR types. The median time to developing anti–MHC class II IgG antibodies was found to be significantly shorter for LVAD recipients with HLA-DR3 (33 days) than for those without this HLA-DR type (103 days) (p = 0.03). By 50 days post-LVAD implantation, 80% of HLA-DR3 individuals had developed anti–MHC class II IgG antibodies compared with only 30% of DR3-negative persons. HLA-DR3 type was also associated with shorter time to developing anti–MHC class I IgG antibodies, although this did not reach statistical significance. No other HLA-DR type significantly influenced onset of anti–MHC antibody production. In additional studies, circulating antiphospholipid antibodies were detected in 7 of 9 (78%) HLA-DR3 LVAD recipients compared with only 5 of 15 (33%) LVAD recipients who were not HLA-DR3 (p < 0.05). Together, these results indicate that inheritance of HLA-DR3 increases susceptibility of LVAD recipients to development of B-cell hyperreactivity.

Clinical consequences of T-cell defects and B-cell hyperreactivity in LVAD recipients

LVAD recipients demonstrate in vivo evidence of defects in cell-mediated immunity: increased prevalence of disseminated candidal infections
We next investigated whether the in vitro defects in cellular immunity identified in LVAD recipients were related to infectious complications in vivo. In a Candidal infection prevalence study, among 78 NYHA class IV heart failure patients listed as UNOS status I and awaiting cardiac transplantation, the presence of LVAD implantation was associated with a significantly increased risk of developing disseminated Candidal infection (defined as positive blood cultures either alone or in association with positive cultures at extravascular sites). By 3 months post-LVAD implantation, 28% of patients with an LVAD had developed disseminated Candidal infection compared with only 3% of those without device implantation (p = 0.0029). Because the risk of developing disseminated Candidal infection persisted throughout the duration of LVAD implantation, with 34% of patients developing an infection by 6 months and 45% of patients by 9 months, this strongly argued that the risk for Candidal infection was the presence of the implanted device rather than any possible effects of surgery. Moreover, no cases of disseminated fungal infection were observed in an additional 425 consecutive patients undergoing cardiac bypass surgery at our institution over the past 12 months by the surgical team principally involved in LVAD implantation.

The clinical consequences of LVAD-related immune dysfunction, particularly disseminated fungal infections, are very serious complications of device implantation. To prevent induction of defects in host immunity and limit such infectious complications, novel strategies need to be developed. In contrast to HIV-1 disease, where the etiology of the severe immune dysfunction is multifactorial, the immune defects accompanying LVAD implantation appear to be far more limited and may, therefore, be more amenable to reversal by therapeutic intervention. One potential approach to prevent AICD is the use of cyclosporine A or FK506, two drugs that inhibit mRNA transcription of FasL after T-cell activation through the TCR complex [24]. We are currently evaluating these and other approaches to reduce the abnormal immune activation present in LVAD recipients.

The presence of IgG anti–MHC class I antibodies increases waiting time to cardiac transplantation
Antibodies in the serum of a cardiac allograft recipient, which are directed against donor HLA class I MHC antigens, constitutively expressed by allograft endothelium portend a significant risk for early graft failure (ie, within the first 24 to 48 hours) and poorer patient survival as a result of complement-mediated humoral rejection [25–27]. Because T lymphocytes constitutively express MHC class I antigens, the presence of preformed lymphocytotoxic antibodies, particularly of IgG isotype, detected in a routine T-cell crossmatch is considered a contraindication to solid organ transplantation [25]. To identify patients at high risk of having a positive donor-specific crossmatch, cardiac transplantation candidates are screened for anti–MHC class I antibodies reactive with lymphocytes from a panel of volunteers representative of the major HLA allotypes, collectively referred to as measurements of panel-reactive antibodies (PRA). Because patients with high PRA levels are considered to be "sensitized" to various alloantigens and require donor-specific cross-matches before transplantation at our institution, we investigated the effects of IgG anti–MHC class I antibodies on waiting time to cardiac transplantation. As expected, LVAD patients with IgG anti–MHC class I antibodies had a significantly longer waiting time than those without these antibodies (175 vs 90 days, p = 0.009) [28]. In contrast, the presence of IgG anti–MHC class II antibodies did not affect the waiting time to transplantation (139 vs 114 days, p = 0.50).

The presence of IgG anti–MHC class II antibodies is a risk factor for high-grade cellular rejections posttransplantation
As shown in Table 1, the presence of IgG anti–MHC class II antibodies detected at the time of transplantation was highly predictive of early high-grade cellular rejection in the posttransplant period [29]. The median time for a high-grade rejection was 70 days for patients positive for IgG anti–MHC class II antibodies. In contrast, the actuarial freedom from rejection never fell below 50% in more than 1,700 days of follow-up for patients without IgG anti–MHC class II antibodies (odds ratio > 24.3, p = 0.006). The presence of IgG anti–MHC class I antibodies was also a moderate risk factor for a high-grade rejection; however, this was not statistically significant (p = 0.08). Additionally, neither the presence of IgM anti–MHC class I nor IgM anti–MHC class II antibodies at the time of transplant influenced the time to a high-grade cellular rejection (p = 0.94 and p = 0.79, respectively). By Cox proportional hazard modeling for multivariable analysis, the only risk factors identified to predict an early high-grade cellular rejection were the presence of pretransplant IgG anti–MHC class II antibodies (p = 0.018) and, to a lesser extent, IgG anti–MHC class I antibodies (p = 0.086). None of the other variables tested in this analysis was predictive of rejection in LVAD recipients, including T-cell PRA, matching at the HLA-DR, -B, or -A loci, ischemic time, or donor age. Additionally, those patients with IgG anti–MHC class II antibodies at the time of transplantation had higher cumulative annual rejection frequencies than those without these antibodies (0.846 vs 0.169 high-grade rejections per patient year of follow-up). Among the demographic and immunologic variables examined, including the other antibody types, only the presence of pretransplant IgG anti–MHC class II antibodies was predictive of a higher cumulative annual rejection frequency, p = 0.002.


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  		Table 1. Influence of Preformed Anti–HLA Antibodies in Cardiac Allograft Recipients at Risk for Sensitization (n = 88) on Cumulative Annual Rejection Frequency Posttransplantation

 
The mechanism by which the presence of pretransplant IgG anti–MHC class II antibodies relates to the posttransplant development of earlier and more frequent high-grade cellular rejections remains conjectural at present. Recent cumulative evidence has emerged that the indirect pathway of CD4 T-cell activation plays a major role in acute and chronic cardiac allograft rejection, due to continuous shedding of donor alloantigenic HLA peptides and their processing by host antigen-presenting cells (APCs) such as macrophages and B-cells. Acute cardiac cellular rejection is accompanied by the appearance both in the circulation and in the allograft of recipient T-cells reactive with donor HLA-DR peptides presented by self-APCs [30]. Primary rejections appear to be invariably accompanied by indirect recognition of a dominant HLA-DR allopeptide [31, 32], whereas recurrent rejections appear to be accompanied by intermolecular spreading and T-cell recognition of multiple donor HLA-DR alloantigenic determinants [32]. Similar patterns of progressive intraand intermolecular HLA-DR epitope spreading can be detected in cardiac transplant recipients developing accelerated transplant-related coronary artery disease [33]. This diversification of the immune response has been postulated to be a result of activation of antigen-specific B-cells by soluble HLA-DR molecules, and the subsequent efficient presentation of multiple HLA-DR allopeptides by self B-cells to CD4 T-cells [34–36]. Therefore, the relationship between recurrent high-grade cellular rejections and preexisting IgG anti–MHC class II antibodies documented in this study may in fact indirectly reflect the presence in sensitized cardiac transplantation candidates of circulating memory B-cells with reactivity to allogeneic HLA-DR molecules.

Therapeutic interventions for B-cell hyperreactivity

A regimen of intravenous immunoglobulin together with intravenous cyclophosphamide is superior to plasmapheresis for reduction of allosensitization
Recent studies have suggested that pooled human intravenous immunoglobulin (IVIg) is an effective modality to reduce allosensitization [37–41]. Postulated mechanisms include the presence in IVIg of antiidiotypic antibodies [42–44], antibodies against membrane-associated immunologic molecules such as CD4 or CD5 [45, 46], or soluble forms of HLA molecules [47]. We investigated the effects of IVIg on serum reactivity to HLA class I molecules in LVAD recipients, and compared these effects to plasmapheresis, an alternative modality for reduction of alloreactive antibodies [48].

We first evaluated the efficacy of monthly IVIg courses, at 2 g/kg, together with monthly infusions of IV cyclophosphamide (0.5 to 1.0 g/m2), on reduction of reactivity of circulating IgG antibodies for allogeneic HLA class I molecules. Data were obtained from 16 patients who received one to three monthly courses of IVIg (total, 28 courses). Each course of IVIg was evaluated as an independent event, and the effects of each IVIg course on IgG anti-HLA class I antibodies during the ensuing 4 weeks were analyzed. Within 1 week after infusion of IVIg in four divided daily doses, the reactivity of circulating IgG antibodies for allogeneic HLA class I molecules was reduced by a mean of 33% (range, 14% to 52%) (p = < 0.01). This was the maximal level of reduction in alloreactivity during the 4 weeks post-IVIg infusion, with the efficacy of IVIg progressively decreasing by the end of week 4 to a mean reduction in alloreactivity of 8% ± 7%. Sequential courses of IVIg did not cause an additive effect on reduction of reactivity of circulating IgG antibodies with allogeneic HLA class I molecules. Each course resulted in a similar level of reduction in alloreactivity compared with baseline, with mean decreases of 38%, 36%, and 35% accompanying first, second, and third courses of IVIg, respectively. Six of the 16 highly sensitized patients were found to be resistant to treatment with IVIg at 2 g/kg, with a mean reduction of only 4% in reactivity of circulating IgG antibodies with allogeneic HLA class II molecules per treatment course in this group. These patients were subsequently treated with one to two courses of high-dose IVIg therapy, 3 g/kg in four divided daily doses. In each patient treated, high-dose IVIg therapy reduced reactivity of circulating IgG antibodies with allogeneic HLA class I molecules. Alloreactivity in this group was reduced by a mean of 20% (range, 16% to 24%) per treatment course (p < 0.05).

We next compared the effects of IVIg (2 g/kg) with plasmapheresis on reduction of reactivity of circulating IgG antibodies with allogeneic HLA class I molecules in LVAD recipients. Four sensitized patients received one to two monthly courses of plasmapheresis, administered two to three times per week (total, six courses). Reactivity of circulating IgG antibodies with allogeneic HLA class I molecules was not significantly reduced within the first 2 weeks after initiation of plasmapheresis. Maximal reduction in alloreactivity, 38% ± 11%, occurred by the fourth week of plasmapheresis. These results show that IVIg has earlier onset of action, and greater efficacy, in reducing IgG anti-HLA alloreactivity compared with plasmapheresis.

Therapy with IVIg together with IV cyclophosphamide shortens the waiting time to cardiac transplantation in sensitized patients
We next investigated whether treatment with IVIg (2 g/kg) together with IV cyclophosphamide (0.5 to 1.0 g/m2) to reduce alloreactivity in sensitized recipients impacted on waiting time to transplantation. The first three highly sensitized LVAD recipients to receive desensitization therapy had unsuccessfully been waiting for cardiac transplantation for a mean of 303 ± 25 days before the onset of therapy as a result of repeated positive donor-specific cross-matches (mean, 33 days; range, 24 to 43 days). After initiation of IVIg/cyclophosphamide therapy, with or without additional immunodepletion using plasmapheresis, all patients obtained negative donor-specific cross-matches and were successfully transplanted in a mean duration of 99 ± 8 days. On the basis of these results, a formal protocol was established to initiate monthly courses of IVIg therapy (2 g/kg) with IV cyclophosphamide after initial detection of allosensitization. The duration from listing to cardiac transplantation was then compared between 28 sensitized patients who did not receive IVIg treatment and 16 sensitized patients who received one to two courses of IVIg (2 g/kg) together with IV cyclophosphamide after detection of anti–HLA class I IgG antibodies. None of these patients received additional plasmapheresis. Whereas the mean duration to cardiac transplantation was 7.1 months (rang, 0.2 to 17.9 months) in patients with IgG antibodies against HLA class I molecules, this was significantly reduced to 3.3 months (range, 0.3 to 6.2 months) in sensitized recipients receiving one to two courses of IVIg (2 g/kg) (p < 0.05). No patient in either group was transplanted across a positive donor-specific IgG T-cell crossmatch. This duration was similar to the waiting time to transplantation in 27 unsensitized patients (3.1 months; range, 0.3 to 10.7 months).

Posttransplant intravenous cyclophosphamide pulse therapy in sensitized cardiac transplant recipients reduces immunologic markers of alloreactivity and prolongs rejection-free interval and decreases cumulative rejection frequency
The posttransplant induction of immunologic markers of allograft rejection were next compared in sensitized cardiac allograft recipients who were treated with cyclosporine/steroid–based triple immunosuppressive regimens incorporating either intravenous cyclophosphamide pulses or oral mycophenolate mofetil. In comparison with mycophenolate mofetil, treatment for 4 to 6 months with intravenous pulses of cyclophosphamide protected against IL2-receptor–positive T-cell outgrowth from biopsy sites during the first posttransplant year (p < 0.01 by regression analysis). Moreover, cyclophosphamide prevented the posttransplant induction of IgG antibodies against HLA class II, but not class I, antibodies (defined as increase by > 10% above pretransplant values). Whereas 9 of 16 (56%) mycophenolate mofetil–treated patients produced increased levels of anti–HLA class II IgG antibodies, only 2 of 16 (13%) cyclophosphamide-treated patients showed an increase in anti–HLA class II IgG antibodies (p = 0.009). Overall, only 4 of 26 (15%) cyclophosphamide-treated patients developed one or more high-grade rejections within the first posttransplant year, compared with 22 of 48 (46%) patients treated with mycophenolate mofetil (p = 0.009, Table 2). Cyclophosphamide treatment had the same effect on sensitized recipients with either preformed IgG anti–HLA class I antibodies (p = 0.02) or class II antibodies (p = 0.04). Moreover, treatment with cyclophosphamide reduced the cumulative annual rejection frequency by 63%, from 0.94 rejections/per year for sensitized patients treated with mycophenolate mofetil to 0.35 rejections per year (p = 0.03). The latter value is within the same range as the annual rejection frequency in nonsensitized patients at our institution. These data indicate that posttransplant use of intravenous cyclophosphamide in sensitized patients is superior to mycophenolate mofetil at preventing recipient T-cell and B-cell responses to donor HLA class II alloantigens.


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  		Table 2. Intravenous Pulse Therapy With Cyclophosphamide Is Superior to Mycophenolate Mofetil for Reduction of Cumulative Annual Rejection Frequency in Sensitized Cardiac Allograft Recipients (48)

 
Safety profile of pre- and posttransplant immunosuppressive therapy in sensitized cardiac allograft recipients
Treatment with intravenous cyclophosphamide has proved to be extremely safe. The incidence of CMV disease (defined as clinical disease together with virologic culture confirmation) was lower in cyclophosphamide-treated patients (3 of 26, 12%) than in those treated with mycophenolate mofetil (10 of 54, 19%). No other viral, bacterial, or fungal infections were seen in patients treated with cyclophosphamide. Intravenous pulse therapy with cyclophosphamide was frequently (> 80%) accompanied by transient nausea and vomiting, which responded to antiemetic therapy. Mesna was coadministered with cyclophosphamide, and may have contributed to the absence of any cases of hemorrhagic cystitis. No malignancies have developed after 540 patient-months of follow-up (range of follow-up per patient, 6 to 38 months). Intravenous immunoglobulin therapy was associated with clinical manifestations of immune complex disease in four of 27 (15%) monthly courses, as evidenced by fevers, arthralgias, and maculopapular rashes. Reversible renal insufficiency (defined as > 50% increase in serum creatinine level) occurred in four cases, all of which resolved spontaneously over the ensuing 3 weeks postinfusion.

Comment

The unquestionable clinical success of left ventricular assist device (LVAD) implantation has, nevertheless, been accompanied by complications arising from interactions between the implanted biomaterial and the host immune system. The aberrant state of monocyte and T-cell activation resulting from these host/device interactions is accompanied by two parallel processes: (1) selective loss of Th1 cytokine producing CD4 T-cells through activation-induced cell death; and (2) unopposed activation of Th2 cytokine producing CD4 T-cells resulting in B-cell hyperreactivity and dysregulated immunoglobulin synthesis through Th2 cytokines and heightened CD40 ligand–CD40 interactions. The net results of these events is that, on the one hand, the LVAD recipient develops progressive defects in cellular immunity and is at increased risk of serious infection, and on the other hand, is more likely to develop allosensitization, posing a significant risk to successful transplant outcome. Intravenous immunoglobulin therapy is an effective and safe modality for sensitized LVAD recipients awaiting cardiac transplantation, reducing serum anti-HLA alloreactivity and shortening the duration to transplantation. The therapeutic and safety profile of IVIg would appear to be superior to plasmapheresis. Immunosuppression incorporating intravenous cyclophosphamide before and after transplantation is safe and highly effective in sensitized LVAD recipients of cardiac transplantation. When used after transplantation as part of triple immunosuppressive regimens, cyclophosphamide is superior to mycophenolate mofetil in reducing episodes of allograft rejection in these patients. Because these immune dysfunctions appear to be related to the effects of excessive biomaterial associated T-cell activation, future efforts will need to be directed at either altering the physical properties of the materials interacting with the host circulation or pharmacological intervention aimed more selectively at inhibiting T-cell activation.

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