Multiple sclerosis: doubling down on MHC.

Human leukocyte antigen (HLA)-encoded surface molecules present antigenic peptides to T lymphocytes and play a key role in adaptive immune responses. Besides their physiological role of defending the host against infectious pathogens, specific alleles serve as genetic risk factors for autoimmune diseases. For multiple sclerosis (MS), an autoimmune disease that affects the brain and spinal cord, an association with the HLA-DR15 haplotype was described in the early 1970s. This short opinion piece discusses the difficulties of disentangling the details of this association and recent observations about the functional involvement of not only one, but also the second gene of the HLA-DR15 haplotype. This information is not only important for understanding the pathomechanism of MS, but also for antigen-specific therapies.

HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

Is multiple sclerosis progression associated with the HLA-DR15 haplotype?

BACKGROUND: The prevalence of multiple sclerosis is associated with the major histocompatibility complex class II DR15 haplotype HLA-DRB1*15:01∼HLA-DRB5*01:01. OBJECTIVE: To assess whether multiple sclerosis progression is associated with the main susceptibility haplotype HLA-DRB1*15:01∼HLA-DRB5*01:01. METHODS: Patients (n = 1230) and healthy controls (n = 2110) were genotyped for HLA-DRB1 and HLA-DRB5. The baseline Expanded Disability Status Scale (EDSS) score was determined and patients were followed for at least 3 years. RESULTS: After follow-up of the consecutive cohort 349 patients were classified as having clinical isolated syndrome and 881 patients as having multiple sclerosis. The susceptibility allele HLA-DRB1*15:01 was more frequent in clinical isolated syndrome (odds ratio 1.56) and multiple sclerosis (odds ratio 3.17) compared to controls. HLA- DRB1*15:01 was the only enriched HLA-DRB1 allele in multiple sclerosis patients. Comparison of clinical characteristics between HLA-DRB1*15:01∼HLA-DRB5*01:01 negative and positive patients with multiple sclerosis showed that baseline EDSS score, disease duration and frequency of the category secondary progressive multiple sclerosis with relapse were increased in the HLA-DRB1*15:01∼HLA-DRB5*01:01 positive group. CONCLUSION: The study confirmed HLA-DRB1*15:01 and HLA-DRB5*01:01 as the main susceptibility alleles and showed weak indirect evidence for a role in progression of the disease.

A rare ancestral HLA-DRB1(∗)15:01̃DQB1(∗)02:01 haplotype and its reversion in the same Western European family.

The HLA-DR and -DQ loci are close neighbors on chromosome 6 that are highly linked. Many common associations between HLA-DR and DQ-alleles are known, normally transmitted as HLA-DR̃DQ haplotypes from one generation to another. Reports of very recent genetic rearrangements between HLA-DR and -DQ are rarely found in the literature. In Europeans haplotypes containing DRB1(∗)15:01, DQB1(∗)02:01, and DQA1(∗)05:01 have not been reported before. We report the finding of the rare HLA haplotype A(∗)24:02̃C(∗)07:02̃B(∗)07:02̃MICA(∗)008:01̃DRB5(∗)01:01̃DRB1(∗)15:01̃DQA1(∗)05:01̃DQB1(∗)02:01̃DPB1(∗)04:01 in a German stem cell donor with East Frisian ancestry. Our observation suggests a rare ancestral recombination between the DR and DQ loci. In order to investigate this haplotype, we typed 50/74 members of the family encompassing four generations for HLA classes I and II by serological and molecular methods. The rare haplotype was identified in 12 heterozygous carriers. Furthermore, we identified and further characterized a putative crossing over event resulting in its reversion to a common haplotype.

High B-cell activating factor is not associated with worse 3-year graft outcome in blood group-incompatible kidney transplantation with rituximab induction.

B cells and their regulation by B-cell activating factor BAFF are of growing interest in kidney transplantation (KTx). There is evidence that high serum (s) BAFF leads to increased allosensitization and impaired long-term graft function. We prospectively investigated sBAFF, peripheral blood lymphocytes (PBL), and donor-specific HLA antibodies (DSA) in patients after ABOi with B-cell depleting rituximab induction treatment and compared them to a group of blood group-compatible (ABOc) living donor kidney recipients. Twelve patients after ABOi and 18 after ABOc were included. After rituximab treatment prior to ABOi, B cells remained significantly lower 1 year after KTx (1.2% (0.0-17.8) compared to ABOc of 8.6% (2.8-35.0), p = 0.0004, and also BAFF-R expression was significantly lower in ABOi (p < 0.006). sBAFF remained elevated 1 year post-Tx compared to ABOc (3615 ± 1800 vs. 1394 ± 493 pg/mL, p < 0.004). Kidney function was not significantly different between both groups after 1, 2, and 3 years. The use of rituximab in ABOi together with maintenance immunosuppression leads to significant elevation of sBAFF and lowering of B-cell numbers for more than 1 year, and this does not correlate with worse 3-year graft outcome.

HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope.

BACKGROUND & AIMS: HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. METHODS: Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. RESULTS: The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. CONCLUSIONS: The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines.

Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor-infiltration of naturally occurring CD8(+) T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire alpha-fetoprotein (AFP), glypican-3 (GPC-3), melanoma-associated gene-A1 (MAGE-A1) and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1) proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8(+) T-cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected interferon-γ (IFN-γ)-producing CD8(+) T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8(+) T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early-stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8(+) T cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8(+) T-cell proliferation but did not restore IFN-γ-production. CONCLUSION: Naturally occurring TAA-specific CD8(+) T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8(+) T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8(+) T-cell responses.

HLA-DR15-derived self-peptides are involved in increased autologous T cell proliferation in multiple sclerosis.

The HLA-DR15 haplotype confers the largest part of the genetic risk to develop multiple sclerosis, a prototypic CD4+ T cell-mediated autoimmune disease. The mechanisms how certain HLA-class II molecules functionally contribute to autoimmune diseases are still poorly understood, but probably involve shaping an autoimmune-prone T cell repertoire during central tolerance in the thymus and subsequently maintaining or even expanding it in the peripheral immune system. Self-peptides that are presented by disease-associated HLA-class II molecules most likely play important roles during both processes. Here, we examined the functional involvement of the HLA-DR15 haplotype in autologous proliferation in multiple sclerosis and the contribution of HLA-DR15 haplotype-derived self-peptides in an in vitro system. We observe increased autologous T cell proliferation in patients with multiple sclerosis in relation to the multiple sclerosis risk-associated HLA-DR15 haplotype. Assuming that the spectrum of self-peptides that is presented by the two HLA-DR15 allelic products is important for sustaining autologous proliferation we performed peptide elution and identification experiments from the multiple sclerosis-associated DR15 molecules and a systematic analysis of a DR15 haplotype-derived self-peptide library. We identify HLA-derived self-peptides as potential mediators of altered autologous proliferation. Our data provide novel insights about perturbed T cell repertoire dynamics and the functional involvement of the major genetic risk factor, the HLA-DR15 haplotype, in multiple sclerosis.

Decreased frequency of CD73+CD8+ T cells of HIV-infected patients correlates with immune activation and T cell exhaustion.

Recent studies indicate that murine Tregs highly express the ENTDP1, as well as the 5'-NT and thereby, suppress Teff function by extracellular adenosine production. Furthermore, CD73 seems to play a role as costimulatory molecule for T cell differentiation. In this study, we analyzed the expression of CD73 on peripheral and lymph nodal Teffs and Tregs in a cohort of 95 HIV patients at different stages of disease, including LTNP and ECs. In contrast to murine Tregs, CD73 was only expressed on a small minority (∼10%) of peripheral Tregs. In contrast, we see high expression of CD73 on peripheral CD8(+) T cells. In HIV infection, CD73 is markedly reduced on all Teffs and Tregs, regardless of the memory subtype. On CD8(+) T cells, a positive correlation between CD73 expression and CD4 counts (P=0.0003) was detected. CD73 expression on CD8(+) T cells negatively correlated with HLA-DR (<0.0001) and PD1 (P=0.0457) expression. The lower CD73 expression on CD8(+) T cells was partially reversible after initiation of ART (P=0.0016). Functionally, we observed that CD8(+)CD73(+) T cells produce more IL-2 upon HIV-specific and unspecific stimulation than their CD73(-) counterparts and show a higher proliferative capacity. These data indicate that down-regulation of CD73 on CD8(+) T cells correlates with immune activation and leads to functional deficits in HIV infection.

Four-year allograft survival in a highly sensitized combined liver-kidney transplant patient despite unsuccessful anti-HLA antibody reduction with rituximab, splenectomy, and bortezomib.

Although donor-specific lymphocytotoxic antibodies are regarded as a contraindication for kidney transplantation (KTx), the data available for liver or combined liver or kidney transplantation (cLKTx) are scarce. Here, we report a case of a highly sensitized young man receiving his sixth liver and second kidney graft. Multiple anti-HLA antibodies were present at the time of transplantation. As a result of suspected antibody-mediated graft damage, the patient was treated with rituximab, plasmapheresis, intravenous immunoglobulins, splenectomy, and bortezomib to decrease the antibody production. So far, patient and allograft survival has reached 4 years despite failure to achieve a permanent reduction of anti-HLA antibodies, and particularly nondonor directed antibodies.

HLA-typing, clinical, and immunological characterization of youth with type 2 diabetes mellitus phenotype from the German/Austrian DPV database.

AIM: To characterize the clinical and immunological features of HLA-typed youth with pediatric onset of type 2 diabetes mellitus (T2DM). METHOD: One hundred and seven patients with clinically diagnosed T2DM (aged ≤20 yr at diagnosis) were examined. DNA and serum, obtained after a median diabetes duration of 2.2 (Q1-Q3: 0.8-4.6) yr, were used for centralized HLA-typing and autoantibody (GADA, IA-2A, ZnT8A) measurements. RESULTS: 64.6% of patients were female and median age at diagnosis was 13.8 (Q1-Q3: 11.6-15.4) yr. Patients were obese [median body mass index-standard deviation score (BMI-SDS): 2.6 (2.0-3.1)], 88.0% had a family history of diabetes and 40.2% a migration background. Islet autoantibodies were detected in 16 (15.0%), among which 7 (6.5%) had multiple islet autoantibodies. Autoantibody positive patients had poorer metabolic control than autoantibody negative patients [glycosylated hemoglobin A1c (HbA1c): 8.1 (6.9-10.1) % vs. 6.6 (5.9-8.0) %; p = 0.033], while patients with HLA-DR genetic risk had higher BMI-SDS than those with HLA-DRXX [2.6 (2.4-3.7) vs. 2.4 (1.7-2.9); p = 0.007]. Metabolic syndrome (61.7%), microalbuminuria (13.4%), and retinopathy (3.9%) were diagnosed. Therapies used were lifestyle only (35.5%), oral anti-diabetics (OAD) only (43.3 %), insulin + OAD (15.9%) and insulin only (5.6%). Patients with ß-cell autoimmunity or HLA-DR genetic risk more frequently used insulin than confirmed T2DM patients (50.0 vs. 22.0%; p = 0.037) and less often had diabetic relatives (61.1 vs. 86.0%; p = 0.030). CONCLUSION: T2DM was confirmed in about 90% of patients while about 10% with ß-cell autoimmunity or HLA-DR genetic risk likely had either T1.5DM or 'double diabetes' or an unknown diabetes type.

Immunological properties of extraembryonic human mesenchymal stromal cells derived from gestational tissue.

Mesenchymal stromal cells (MSCs) have been isolated from many tissues, including gestational tissue. To date, a study comparing the properties and suitability of these cells in cell-based therapies is lacking. In this study, we compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory capabilities of human MSCs derived from umbilical cord lining (CL-MSCs), umbilical cord blood (CB-MSCs), placenta (P-MSCs), and Wharton's jelly (WJ-MSCs). Differences were noted in differentiation, proliferation, and migration, with CL-MSCs showing the highest proliferation and migration rates resulting in prolonged survival in immunodeficient mice. Moreover, CL-MSCs showed a prolongation in survival in xenogeneic BALB/c mice, which was attributed to their ability to dampen TH1 and TH2 responses. Weaker human cellular immune responses were detected against CL-MSCs and P-MSCs, which were correlated with their lower HLA I expression. Furthermore, HLA II was upregulated less substantially by CL-MSCs and CB-MSCs after IFN-γ stimulation. MSC types did not differ in indolamine 2,3-dioxygenase (IDO) expression after IFN-γ stimulation. Despite their lower IDO, HLA-G, and TGF-ß1 expression, only CL-MSCs were able to reduce the release of IFN-γ by lymphocytes in a mixed lymphocyte reaction. In summary, CL-MSCs showed the best characteristics for cell-based strategies, as they are hypo-immunogenic and show high proliferation and migration rates. In addition, these studies show for the first time that although immunomodulatory molecules HLA-G, HLA-E, and TGF-ß play an important role in MSC immune evasion, basal and induced HLA expression seems to be decisive in determining the immunogenicity of MSCs.

Comparing individual NK cell activity in vitro.

Natural killer (NK) cells play an important role in the innate immune system by eliminating infected and mutated cells. Their cytotoxic capacities vary markedly among individuals. The cytotoxic activity can be measured in peripheral blood mononuclear cells (PBMCs) using the NK cell-specific target cell line K562. In this chapter, we present a protocol for the standardization and normalization of cell preparation and NK cell cytotoxicity measurement in a (51)Cr-release assay. By following these protocols, it is possible to compare the NK cell activity of numerous-if necessary selected-individuals in vitro.

T cell epitope mapping of JC polyoma virus-encoded proteome reveals reduced T cell responses in HLA-DRB1*04:01+ donors.

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4(+) T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4(+) T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01(+), DRB1*07(+), DRB1*11(+), DRB1*13(+), DRB1*15(+), and DRB1*03(+) individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01(+) individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.

TCR bias and HLA cross-restriction are strategies of human brain-infiltrating JC virus-specific CD4+ T cells during viral infection.

Virus-specific CD4(+) T cells play a central role in control of viral pathogens including JC polyoma virus (JCV) infection. JCV is a ubiquitous small DNA virus that leads to persistent infection of humans with no clinical consequences. However, under circumstances of immunocompromise, it is able to cause an opportunistic and often fatal infection of the brain called progressive multifocal leukoencephalopathy (PML). PML has emerged as a serious adverse event in multiple sclerosis patients treated with the anti-VLA-4 mAb natalizumab, which selectively inhibits cell migration across the blood-brain barrier and the gut's vascular endothelium thus compromising immune surveillance in the CNS and gut. In a multiple sclerosis patient who developed PML under natalizumab treatment and a vigorous immune response against JCV after Ab washout, we had the unique opportunity to characterize in detail JCV-specific CD4(+) T cell clones from the infected tissue during acute viral infection. The in-depth analysis of 14 brain-infiltrating, JCV-specific CD4(+) T cell clones demonstrated that these cells use an unexpectedly broad spectrum of different strategies to mount an efficient JCV-specific immune response including TCR bias, HLA cross-restriction that increases avidity and influences in vivo expansion, and a combination of Th1 and Th1-2 functional phenotypes. The level of combinatorial diversity in TCR- and HLA-peptide interactions used by brain-infiltrating, JCV-specific CD4(+) T cells has not, to our knowledge, been reported before in humans for other viral infections and confirms the exceptional plasticity that characterizes virus-specific immune responses.

Impact of the NK cell receptor LIR-1 (ILT-2/CD85j/LILRB1) on cytotoxicity against multiple myeloma.

The role of different receptors in natural-killer- (NK-) cell-mediated cytotoxicity against multiple myeloma (MM) cells is unknown. We investigated if an enhancement of NK-cell-mediated cytotoxicity against MM could be reached by blocking of the inhibitory leukocyte immunoglobulin-like receptor 1 (LIR-1). Our investigations revealed high levels of LIR-1 expression not only on the NK cell line NK-92, but also on myeloma cells (MOLP-8, RPMI8226) as well as on a lymphoblastoid cell line (LBCL; IM-9). Subsequent cytotoxicity assays were designed to show the isolated effects of LIR-1 blocking on either the effector or the tumor side to rule out receptor-receptor interactions. Although NK-92 was shown to be capable of myeloma cell lysis, inhibition of LIR-1 on NK-92 did not enhance cytotoxicity. Targeting the receptor on MM and LBCL did not also alter NK-92-mediated lysis. We come to the conclusion that LIR-1 alone does not directly influence NK-cell-mediated cytotoxicity against myeloma. To our knowledge, this work provides the first investigation of the inhibitory capability of LIR-1 in NK-92-mediated cytotoxicity against MM and the first functional evaluation of LIR-1 on MM and LBCL.

Elevated serum levels of B-cell activating factor in pediatric renal transplant patients.

BACKGROUND: B-cells are increasingly recognized as important players in alloimmunity. B cell-activating factor (BAFF) and its receptor BAFF-R are essential for B-cell differentiation and survival. Data on BAFF levels in pediatric renal transplant (RT) patients are scarce. OBJECTIVE: It is known from adult data that elevated BAFF levels correlate with an unfavorable outcome in bone marrow and kidney recipients. To analyze this hypothesis in pediatric renal transplant patients we performed a cross-sectional analysis of serum BAFF levels, lymphocyte surface BAFF-R expression, and clinical variables in a cohort of 43 pediatric renal transplant patients. METHODS: We studied serum BAFF, CD19+ B-, and FoxP3+ regulatory T-cells (Tregs) and BAFF-R expression in 43 children 2.9 (0.1-12.4) years after RT on maintenance immunosuppression. Twenty-two healthy children and 19 children with chronic kidney disease stage 5 (CKD5) served as controls. RESULTS: BAFF levels were significantly higher in RT patients than in healthy children (1,435±574 vs 894±189 pg/mL; p<0.0001) whereas numbers of B-cells and Tregs were significantly lower. BAFF-R expression on B-cells was decreased after RT (531±334 vs 707±257 MFI; p<0.005), BAFF inversely correlated with BAFF-R (r=-0.5022, p<0.006), but not with B-cell count. BAFF was elevated in CKD5 patients (1,276±294 pg/mL). In RT patients BAFF was significantly higher in those with eGFR <60 ml/min/1.73m(2) (1,553±447 vs 1,234±323 pg/mL; p=0.02). BAFF levels and BAFF-R expression did not correlate with HLA antibody status, time after transplantation, age or gender of the patients. CONCLUSION: Serum BAFF concentrations were significantly elevated in pediatric RT patients. They correlated with decreased BAFF-R expression on CD19+ B-cells and impaired allograft function. Our findings of a dysregulated BAFF/BAFF-R axis may be of clinical relevance after renal transplantation and therefore underline the importance of further research into BAFF-dependent mechanisms.

Assessment of physiologic natural killer cell cytotoxicity in vitro.

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.