Assessment of humoral and cellular immune responses to SARS CoV-2 vaccination (BNT162b2) in immunocompromised renal allograft recipients.

BACKGROUND: Assessing the composition of immune responses to SARS-CoV-2 vaccines is critical for our understanding of protective immunity, especially for immune compromised patients. The Pfizer (BNT162b2) vaccination showed >90% efficacy in protecting individuals from infection. However, these studies did not examine responses in immunocompromised kidney transplant patients (KT). Subsequent reports in KT have shown severe deficiencies in Spike-specific immunoglobin G (IgG) responses prompting booster vaccinations, but a broader understanding of T-cell immunity to vaccinating is lacking. METHODS: We examined SARS-CoV-2 Spike IgG and CD4+/CD8+ Spike-specific T-cell responses in 61 KT patients maintained on different immunosuppressive protocols (ISP) (Tac + mycophenolate mofetil + prednisone) versus (belatacept + MMF + prednisone) and compared to 41 healthy controls. We also examined cytomegalovirus-cytotoxic T-cell responses (CMV-Tc) in both groups to assess T-cell memory. RESULTS: Our data confirmed poor Spike IgG responses in vaccinated KT patients with both ISP (21% demonstrating Spike IgG 1M post-second dose of BNT162b2 vs. 93% in controls). However, 35% of Spike IgG (-) patients demonstrated CD4+ and/or CD8+ T-cell responses. All but one CMV-IgG+ patient demonstrated good CMV-Tc responses. No differences in T-cell immunity by ISP were seen. CONCLUSION: Immunocompromised KT recipients showed severe defects in humoral and T-cell immune response after vaccination. No differences in immune responses to SARS-CoV-2 Spike peptides were observed in KT patients by ISP post-vaccination. The detection of Spike-specific T-cell immunity in the absence of Spike IgG suggests that vaccination in immunocompromised KT patients may provide partial immunity, although not preventing infection, T-cell immunity may limit its severity.

Cytomegalovirus Immunity After Alemtuzumab Induction in Desensitized Kidney Transplant Patients.

BACKGROUND: Desensitization with IVIG + rituximab combined with alemtuzumab induction gives HLA-sensitized patients an opportunity for successful kidney transplantation. However, it may be associated with a high risk for viral infections due to combined T cell and B cell depletion. METHODS: Anti-cytomegalovirus (CMV) activity was assessed in 280 pretransplant and posttransplant blood samples from 33 desensitized patients who received alemtuzumab induction. CMV-specific CD8+ (CMV-Tc), CD4+ (CMV-Th) T cell activity, and natural killer (NK) cell number were measured by flow cytometry. Anti-CMV IgG was measured by enzyme-linked immunosorbent assay, and CMV DNA by polymerase chain reaction. RESULTS: All 30 CMV sero (+) patients were (+) for CMV-Tc and/or Th predesensitization, while 3 sero (-) patients showed no CMV-T cell activity. CMV-Tc and/or Th became (-) in 50% to 70% of these sero (+) patients at 1 month post-alemtuzumab. However, 75% showed CMV-T cell (+) by 2 months and 95% did so by 3 months post-alemtuzumab. More than 50% of pretranslpant NK cell levels were detected post-alemtuzumab. Anti-CMV IgG levels did not decrease posttransplant in sero (+) patients. Four patients developed CMV viremia with clearance by 1.2 months, which correlated with an increase or appearance of CMV-T cells, even in the sero (-) patient. CONCLUSIONS: CMV-T cell activity, anti-CMV IgG, and NK cell-mediated antibody-dependent cell cytotoxicity were present in aleumtuzumab-treated CMV sero (+) patients. One sero (-) patient developed CMV-T cell responses post-CMV viremia. These results suggest that the IVIG + rituximab desensitization combined with alemtuzmab induction with triple immunosuppression maintenance does not result in prolonged suppression of anti-CMV immunity or increased risk for CMV infection.

Impact of Desensitization on Antiviral Immunity in HLA-Sensitized Kidney Transplant Recipients.

Viral infections represent significant morbidity and mortality factors in kidney transplant recipients, with CMV, EBV, and BKV infections being most common. Desensitization (DES) with IVIg and rituximab with/without plasma exchange followed by kidney transplantation with alemtuzumab induction increased successful transplant rates in HLA-sensitized patients but may represent an increased risk for viral infections due to severe lymphocyte depletion. Here, we report on the posttransplant viral infection status in 372 DES versus 538 non-DES patients. CMV and EBV viremia were significantly lower in DES patients, while BKV viremia was similar. This trend was observed primarily in CMV sero(-), EBV sero(+), and sero(-) patients. No patient developed PTLD. The incidence of BKAN, allograft, and patient survival was similar in both groups. These viral infections were not associated with subsequent allograft rejection which occurred within 6 months after the infection. Conclusions. The IVIg + rituximab desensitization combined with alemtuzumab induction with triple immunosuppression maintenance does not increase the risk for CMV, EBV, and BKV infections. Possible factors include, in addition to posttransplant antiviral prophylaxis and PCR monitoring, presence of memory T cells and antibodies specific to CMV and likely EBV, NK cell-mediated ADCC despite lymphocyte depletion, elimination of EBV and CMV reservoirs by rituximab and alemtuzumab, and use of IVIg with antiviral properties.

Immunomodulatory effects of combination of pooled human gammaglobulin and rapamycin on cell proliferation and apoptosis in the mixed lymphocyte reaction.

BACKGROUND: Multidrug immunosuppressive regimens benefit transplant recipients, reducing side effects and creating synergy between medications with different mechanisms of action. We have shown that pooled human gammaglobulin (intravenous immunoglobulin [IVIG]) inhibits the mixed lymphocyte reaction (MLR) and induces apoptosis primarily in B cells. Rapamycin (RAPA), a potent macrolide immunosuppressant, inhibits B- and T-cell proliferation through G1 cell-cycle blockade and purportedly induces apoptosis. Here we examined the possible synergistic effects of IVIG and RAPA on cell proliferation and apoptosis induction in the MLR. METHODS: MLR was performed with IVIG (0.2-5 mg/mL), RAPA (0.02-40 ng/mL), alone or in combination. Cell proliferation was detected by H-thymidine incorporation, and apoptosis by Annexin V and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling flow cytometry. RESULTS: IVIG or RAPA inhibited cell proliferation in a dose-dependent manner. RAPA (0.02-40 ng/mL) in combination with IVIG (5 mg/mL) significantly augmented the inhibition compared with RAPA alone (70% vs. 34% at 0.2 ng/mL; 90% vs. 76% at 2 ng/mL). Apoptosis was significantly higher in IVIG-treated (5 mg/mL) CD19+ cells and less so in CD3+ cells. However, RAPA (0.2-40 ng/mL) neither induced apoptosis nor altered apoptosis induced by IVIG. CONCLUSIONS: Combined RAPA and IVIG at subtherapeutic concentrations inhibits cell proliferation in the MLR. RAPA neither induces apoptosis nor augments apoptosis induced by IVIG in the MLR. Lower-concentration RAPA (0.2-2 ng/mL) in combination with IVIG (5 mg/mL) versus therapeutic levels (2-50 ng/mL and 10-40 mg/mL, respectively) could represent an effective immunomodulatory drug combination.

Significant reduction of ATP production in PHA-activated CD4+ cells in 1-day-old blood from transplant patients.

BACKGROUND: Global immunosuppression can be measured by assessing adenosine triphospate (ATP) levels in mitogen-stimulated CD4+ T cells. METHODS: We investigated the effect of storage time on ATP levels in 234 blood samples from 18 healthy individuals and 152 transplant patients. The difference between day 0 (<13 hours post-blood draw) and day 1 (24-37 hours) measurements was analyzed and compared with various factors; a subset of samples was also analyzed in 6-hour intervals. RESULTS: The ATP levels were significantly lower on day 1 compared with that on day 0 in healthy individuals (279±159 vs 414±159 ng/mL, P<0.001) and patients (356±209 vs 455±221 ng/mL, P<0.0001). Of the 18 healthy individuals, 17 showed ATP reduction, whereas 192 (89%) of 216 patients did so on day 1 (24.8±24.1%). In the time course analysis, ATP levels decreased with the blood storage time in healthy and patient samples, and the reduction began as early as 7 hours post-blood draw. The reduction rate was significantly higher in patient samples with low day 0 ATP levels compared with samples with moderate or high levels (44.7±31.3% vs 23.2±23.6% or 18.7±15.7%; P<0.001). The reduction rate in patients treated with alemtuzumab induction was slightly higher than that in daclizumab-treated patients (28.8±24.6% vs 21.3±21.3%, P=0.09). CD4+ cell number did not change within 24 hours post-blood draw, but CD4 expression decreased 2.0±2.8% (P<0.05). CONCLUSIONS: The ATP levels are significantly lower in 1-day-old blood compared with fresh blood, suggesting that fresh blood should be used for assessing the T cell immune function to obtain the most accurate results.

Immunologic parameters and viral infections in patients desensitized with intravenous immunoglobulin and rituximab.

Desensitization with IVIG and rituximab followed by transplantation with alemtuzumab or daclizumab induction is an effective clinical protocol. Here, we examined the effects of this protocol on immune cell number, T cell function by Cylex ImmuKnow®, CMV-specific CD8+ T cell (CMV-Tc) activity, total and viral-specific immunoglobulin levels and viral infections. In 17 highly HLA-sensitized (HS) patients who received desensitization, CD19+ cells were undetectable immediately after desensitization, while other immune cells were unchanged. No alteration in Cylex or CMV-Tc levels was seen. In separate 14 HS patients who were desensitized followed by transplantation, T cell numbers were near zero after alemtuzumab, while NK cell reduction was minimal. Early B cell recovery was not a risk for antibody-mediated rejection. Total IgG, IgM, and IgA remained in the normal range up to 12.6 months post-transplant, and CMV IgG level did not change. CMV-Tc activity was eliminated post-transplant in some patients, but recovered by 4 months post-transplant. None of them developed CMV infection. In conclusion, IVIG-rituximab-desensitization does not significantly alter T cell function pre-transplant, or reduce Ig levels below the normal range post-transplant. Although post-transplant induction therapy is associated with a transient depletion of viral-specific CD8+ memory cells, it does not increase risks for viral infections.

In vitro effects of everolimus and intravenous immunoglobulin on cell proliferation and apoptosis induction in the mixed lymphocyte reaction.

Targeting multiple pathways in the activation of alloimmune responses by multi-drug immunosuppressive regimens with complementary mechanisms of action enhances allograft survival and improves quality of life, owing to the reduction of adverse drug effects. In this report we investigated the effect of the combination of everolimus and intraveneous immunoglobulin (IVIG) on cell proliferation and apoptosis induction in human two-way mixed lymphocyte reaction (MLR). Everolimus alone (0.1-50 ng/ml) and IVIG (1-10 mg/ml) alone inhibited cell proliferation in a dose-dependent manner (16.4-67.2% and 12.1-66.3% inhibition, respectively). The inhibition by everolimus was not enhanced in the presence of 1 mg/ml IVIG. Addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG, but only by 10-27%. Addition of 0.1 and 1 ng/ml everolimus did not increase IVIG's inhibitory effects. Apoptosis was significantly higher in IVIG (5 mg/ml)-treated CD19+ cells and less so in CD3+ cells as assessed by Annexin V and TUNEL assays. However, everolimus (0.1-50 ng/ml) did not induced apoptosis or alter apoptosis induced by IVIG. These results suggest that everolimus is a potent inhibitor of immune cell proliferation but does not act additively or synergistically with IVIG when analyzed in this in vitro system.

Intracellular IFNgamma production in CD3 negative cells exposed to allo-antigens is an indicator of prior sensitization.

BACKGROUND: Sensitization to HLA antigens (Ags) is a significant obstacle to kidney transplantation and risk factor for antibody-mediated rejection (AMR). Current screening methods to assess HLA Ag exposure include various antibody assays. However, tools to accurately measure cell-mediated immunity to allo-Ags in a clinical setting are lacking. Here we report on an intracellular cytokine flow cytometry (CFC) assay that detects intracellular gamma-interferon (IFNgamma) production in non-T cell populations (CD3-) that appears to assess sensitization from previous allo-Ag exposure. METHODS: Blood from 106 highly-HLA sensitized (HS) patients (pre-, post-IVIG-treatment [Rx] and/or post-transplant) and 14 3(rd) party normal controls (3(rd)N) were incubated with donor or 3(rd)N peripheral blood mononuclear cells (PBMCs), and IFNgamma+/CD3- cells were enumerated. RESULTS: The percentage of IFNgamma+ cells in CD3- cells without stimulation in pre-IVIG-Rx HS patients was similar to normals, but significantly increased with incubation with donor and/or 3(rd)N PBMCs. Reactivity in normals was minimal. Reactivity was higher in HS females than HS males. Normal females with previous pregnancy (PG) showed significantly higher response than females without PG or non-sensitized normal males. Donor-specific reactivity in the CFC assay better correlated with donor-specific B cell crossmatch than total anti-HLA antibody levels or PRA. HS patients who developed AMR post-transplant showed significantly higher reactivity than those without AMR. CONCLUSIONS: The CFC assay measures IFNgamma production in CD3- cells that may indicate a memory response to allo-Ags. This response is limited to HS patients and normal females with previous PG. Patients undergoing AMR show significantly higher reactivity. This assay may represent a novel approach to measurement of allo-sensitization with clinical utility in predicting those at risk for AMR.

Cellular immune responses to cytomegalovirus in renal transplant recipients.

Control of CMV replication depends primarily on anti-CMV T lymphocyte activity. However, the functional T-cell responses to CMV in immunosuppressed solid organ transplant recipients are not well understood. In this study we employed cytokine flowcytometry (CFC) using pooled CMV peptides and viral lysates to detect CMV-specific T-cell responses in 17 healthy controls, 33 stable renal transplant recipients (Tx recipients) and 6 transplant recipients with active CMV infection (CMV(+)). We found that pooled peptides and lysates provide optimal detection of IFN gamma production in anti-CMV CD8(+) and CD4(+) T cells, respectively. In both healthy controls and Tx recipients, CMV-specific T-cell levels strongly correlated with serostatus. Seropositive Tx recipients have significantly higher levels of CMV-specific CD8(+) T-cell responses compared to healthy controls, which may signify an effort to control enhanced viral replication in immunosuppressed Tx recipients. In some individuals, absence of anti-CMV T-cell response may correlate with lack of viral clearance by ganciclovir therapy, even when CMV isolates are not ganciclovir resistant. Thus, monitoring cellular immunity with CFC along with viral load by PCR merits further exploration for identification of patients at the risk of developing CMV disease, tailoring prophylactic and therapeutic decisions and preventing complications.