Profound T-cell depletion with the monoclonal antibody alemtuzumab facilitates reduced maintenance immunosuppression in abdominal and lung transplantation. While the phenotype of the post-depletional T cells has been characterized, little is known about their function. In the present study, global and CMV-specific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow® and T Cell Memory™, Cylex, Columbia, MD) during the first year posttransplant after induction therapy. Recovery of mitogen responses were seen at 2 weeks posttransplantation (65%PHA; 58% Con A), despite the low number of circulating T cells (<2%). These responses declined at 4–5 months (24%PHA; 54% Con A) and were partially reconstituted by 9 months (46% PHA; 73% Con A). CMV-specific responses recovered in 80% of R+ patients as early as 2 weeks posttransplant (n = 5) and 72% of patients had a memory response by 3 months (n = 11). In contrast, only 2 of 5 patients who did not exhibit memory responses pre-transplant (R–) developed transient CMV-specific T-cell responses. Our results show that profound depletion of T cells induced by alemtuzumab spares the functional subset of CMV-specific memory T cells. Conversely, CMV R– patients predepletion may require a prolonged period of prophylaxis.
Immunosuppressive strategies utilizing peri-transplant profound lymphoid depletion have been developed to allow reduced dose maintenance immunosuppression with the hope of reducing toxicities associated with chronic multidrug immunosuppressive therapy and have been suggested to possibly promote a pro-tolerant state (1,2). Several groups have now demonstrated that induction therapy with the humanized anti-CD52 monoclonal antibody results in profound and sustained depletion of T cells peripherally and in secondary lymphoid tissues allowing for reduced dose maintenance immunosuppression in kidney, liver, intestine and pancreas transplantation and most recently in pulmonary transplantation by our group (3–8). However, such T-cell-depleting strategies have raised concern about infectious risk from opportunistic pathogens particularly in pulmonary transplantation where infection remains a significant cause of morbidity and mortality. While recent studies have characterized the residual postdepletional T cells as phenotypically memory-like, little is know of the functional capacity of these cells with the exception that they are capable of initiating alloimmunity (9). Retention of functional memory T cells to environmental pathogens post-depletion would be expected to result in preserved immunity to these pathogens following administration of T-cell-depleting agents.
Cytomegalovirus (CMV) remains a significant cause of morbidity and mortality for lung transplant (LTx) recipients. The incidence of CMV infection and disease is higher in LTx recipients than other solid organ transplant patients with a reported incidence of 54–92% in patients without anti-CMV prophylaxis (10,11). While there are several identifiable risks factors for CMV disease, the most relevant factor is the donor (D) and recipient (R) sero-status with D+/R– being at the highest risk (11). The role of the cellular immune response in controlling CMV infection following induction therapy is not well understood. The in vitro functionality of CD4+ T cells has previously been shown to be a dependable measurement for immunocompetence (12–14). Global and antigen specific T-cell function can be measured in peripheral blood by both in vitro stimulation of the cells with mitogen (Con A and PHA) and specific antigen.
In the current study, we longitudinally investigated the recovery of mitogen T-cell and CMV-specific responses in the peripheral blood of lung transplants who received induction therapy with alemtuzumab utilizing the Cylex ImmuKnow® and T Cell Memory™ cell mediated immune function tests, respectively. We examined for both de novo T-cell responses in those patients naïve to CMV as well as retention and/or recovery of memory responses. We demonstrate that T-cell depletion with alemtuzumab spares the functional subset of CMV-specific memory T cells and that significant recovery of global mitogen responses are seen as early as 2 weeks postdepletion.
Patients and Methods
Peripheral blood from 23 lung transplant recipients at the University of Pittsburgh were obtained prior to transplant and at their subsequent clinical follow-up. Seven patients were seropositive and received a lung from a seropositive donor, (R+/D+), seven were seropositive and received a lung from a seronegative donor (R+/D), five were seronegative and received a lung from a seropositive donor (R−/D+) and four were seronegative and received a lung from a seronegative donor (R−D−). Human volunteer participation in these studies was according to HHS and HIPAA regulations and approved by the Institutional Review Board at the University of Pittsburgh. Informed consent was obtained from each patient participating in this study. All lung transplant recipients received universal prophylaxis with Valganciclovir (450–900 mg) orally every day. The dosage of Valganciclovir was adjusted for renal function. The patients also underwent weekly monitoring with pp65 antigenemia while on prophylaxis.
Cell-mediated global and CMV-specific assays
Sodium heparin anticoagulated whole blood samples were collected during routine or unscheduled clinic or in-hospital visits under approved institutional IRB procedures. Immune responses were measured using the Cylex® ImmuKnow® and T Cell Memory™ assay according to the manufacturer's package insert (15,16). Briefly, anticoagulated whole blood was diluted with sample diluent, added to wells of a 96-well microtiter plate and incubated for 15–18 h with PHA, Con A or a CMV-containing cell lysate in a 37° C, 5% CO2 incubator. The following day, CD4+ (ImmuKnow®) or CD3+ (T Cell Memory™) T-cells were positively selected within the microwells using magnetic particles coated with antihuman CD4 and antihuman CD3 monoclonal antibodies (Dynabeads, Dynal, Oslo Norway) and a strong magnet (Cylex Magnet tray 1050, Cylex Inc. Columbia, MD), washed to remove residual cells and lysed to release intracellular ATP. Released ATP was measured using luciferin/luciferase and a luminometer (Berthold, Knoxville, TN or Turner Biosystems, Sunnyvale, CA). Assay precision demonstrated an overall pooled percent coefficient of variance of 11.6%. A patient's level of immune response was assessed based on the amount of ATP expressed in nanograms per milliliter. A positive response is determined by comparing the ATP concentration (ng/mL) for the stimulated sample to the nonstimulated sample. A three fold increase over the background is considered a positive response for CMV. The CMV-specific memory response was considered positive if the ATP was at least threefold or higher than the unstimulated background and the overall counts were ≥10ng/mL ATP.
PHA and Con A responses
The impact of alemtuzumab induction therapy on the preservation of global (PHA and Con A) T-cell responses was assessed from the time of lung transplantation (LTx) to 1 year following transplantation in 23 patients. Because the pretransplant range of ATP values for patients were quite heterogeneous (PHA: 264–924ng/mL; Con A: 59–607 ng/L), the recovery of mitogen responses was calculated as a percentage of baseline for each patient at each time point and ultimately shown as the mean for all patients.
Recovery of PHA and Con A response were 58% and 65%, respectively, of pretransplant values at 2 weeks post-LTx (Figure 1A and B). Despite a very low number of circulating T cells in the first 3 months postdepletion therapy, the PHA and Con A responses were maintained at 40–60% of pre-Tx levels. Four to 6 months post-LTx, PHA responses drop to 21% of pretransplant levels while the Con A responses remained at 60%. However, by 6–9 months, the CD4+ T-cell responses to PHA recovered to 50% of pre-Tx levels, but were still lower than the CD3+ T cell responses to Con A (70%). Mitogen responses to PHA and Con A did not differ between LTx patients who were CMV seropositive or seronegative prior to transplantation (data not shown).
CMV-specific T-cell responses
In R+/D+/− patients, CMV-specific memory responses could be detected as early as 2 weeks post-LTx in 80% of the patients tested (Figure 2). In contrast, no CMV-specific T-cell memory responses were detected during the same period in recipients who were CMV seronegative (R−) prior to transplantation. Moreover, CMV T-cell memory responses were detected in 72% of R+/D+/− patients during the first 3 months posttransplantation (8/11 patients; 11/16 samples tested). CMV-specific responses developed in two of five R−/D+ patients by 3 months post-transplantation (on days 44 and 87 post-LTx), but these responses tended to be weaker in magnitude and only transiently detected during the first 3 months (Figure 2). None of R−/D− patients exhibited CMV specific memory responses. Longitudinal assessment of mitogen and CMV-specific responses are shown in Figure 3. CMV T-cell memory responses were detected as early as 2 weeks post-transplant in R+/D+ individuals and were generally maintained throughout the first year, as exemplified by the individual patient profile shown in Figure 3A. This individual had a robust CMV T-cell response throughout the first year with the exception of two time points within the first 6 months, which was a pattern seen in most R+/D+ individuals. Interestingly, mitogen responses also tended to be lower at time points when CMV responses were suppressed. A second profile is shown for a R+/D− patient in Figure 3B. During the post-Tx follow-up, this patient exhibited global and CMV-specific responses that recovered to pre-Tx values at 330 days post-Tx. In contrast, a R−/D+ patient did not have a detectable CMV-specific response until 3 months and this response was transient (Figure 3C). During the same time period, we could detect PHA and Con A responses supporting the concept that this patient was immunocompetent but could not exhibit a CMV-specific primary response following T-cell depletion treatment. (16). The values for all patients who were positive pretransplant were higher than post-Campath depletion (range 20–60 ng/mL ATP) and posttransplant (10–20 ng/mL; as depicted in Figure 3A–C).
In this study, we have shown the functional recovery of CMV-specific memory T cells in lung transplant recipients during the early postinduction period with a potent anti-lymphoid preparation (alemtuzumab). Previously, Pearl et al. have reported that the dominant type of T cells following antibody-mediated T-cell depletion had a phenotype consistent with a memory phenotype CD3+CD4+CD45RA−CD62L−CCR7− (9). Our findings support the concept of preservation of memory T cells following aggressive depletion and provide functional assessment of antigen specific T cells.
Despite a rapid decline in the number of T cells in peripheral blood after a single dose of the humanized CD52-specific monoclonal antibody alemtuzumab, we observed global immunocompetence as reflected by responses to mitogens (PHA and Con A) at significant levels within 2 weeks (Figure 1A and B). Overall, the levels of ImmuKnow testing (PHA stimulation) were between 100 and 300 ng/mL ATP, similar to the previously published levels in other stable, solid organ transplant recipients (15,16).
We have previously shown that alemtuzumab induced a more prolonged CD3+ T cell depletion than thymoglobulin with minimal recovery of lymphoid cells within the first 3 months (8). Nevertheless, greater than 50% of patients who were CMV sero-positive pre-transplantation (R+) exhibited CMV-specific memory responses during this early post-T-cell depletion period. The majority of the R+ group displayed memory responses by 9 months post-LTx. In contrast, only a few of the CMV naïve LTx recipients (R−) with T-cell depletion developed transient CMV-specific memory T cells by 3 months. These LTx recipients required more than 9 months to develop stable CMV-specific memory T cells after primary CMV infection.
Primary CMV infection in lung transplant recipients is associated with increased mortality. Currently, preemptive as well as universal prophylaxis with ganciclovir or valganciclovir are employed for the prevention of CMV disease in lung transplantation. The optimal duration of prophylaxis with ganciclovir or valganciclovir in lungs is not well known. However, from earlier experience in lung transplant recipients, there is a clear association between duration of ganciclovir prophylaxis and incidence of CMV disease. Incidence of CMV disease dropped from 85% after 2 weeks of ganciclovir prophylaxis to 38% after 3 months of ganciclovir therapy (11,17–19). In one study, freedom from CMV infection or disease was noted in 95% of patients receiving valganciclovir for 270 days (20). While these approaches have been shown to be effective in reducing CMV disease, toxicities and drug resistance associated with treatment remain a concern (11,17,20–24).
Our laboratory had previously shown that seropositive R+/D(±) LTx recipients had a higher level of T-cell memory responses as compared to seronegative (R−) recipients (13). This phenomenon is likely due to pre-existing memory prior to transplantation and boosting of the response by donor CMV antigens present in the graft. Additionally, those patients that exhibit CMV-specific responses (R+) are free from recurrent CMV disease when compared to patients with a persistent lack (R−/D+) of CMV-specific memory following lung transplantation (13).
Shlobin et al. recently reported that despite significant immunosuppression in R−D+ patients, a majority develop durable CMV memory responses after primary CMV infection (25). Our data support these findings and extend them by showing that R−D+ patients develop CMV-specific responses, but that induction therapy can potentially delay this process. Our results also indicate that patients who are seropositive prior to transplant maintain a detectable CMV memory response immediately following induction therapy (Figures 1 and 2A and B).
There are a number of limitations to this study. The overall number of patients was small, especially the number of R−/D+ lung transplant recipients. However, the moderate size of CMV seropositive patients' pre-LTx allowed us to evaluate the recovery of immunocompetence and CMV-specific memory after an aggressive T-cell depletion protocol. Further, investigation with a larger number of patients and more frequent sampling is required to better determine the extent of prophylaxis these patients need.
In summary, our results suggest that the T Cell Memory™ test can be used to identify antigen-specific memory T cells in lung transplant recipients with minimal circulating T cells following aggressive T-cell depletion. The applications of these results in stratifying the patients at risk of acute CMV disease, and hence initiation or termination of CMV prophylaxis, remains to be determined in larger studies.