ICI 2016_Abstract Book

852

cytotoxicity detection assay. We stained three different cancer cell

lines (one of which had intact MHC receptors) with three different

dyes and incubated them in the same well with Effector to Target

ratios that match one cell line per well. The results obtained show

that we can simultaneously detect the cytolytic effect of NK cells on

three different target cell types using only a third of the effector cells

as previously required. Also, the data show that control target cells

with MHC receptors are not susceptible toNK killing.

Four color T- and B cell ELISPOT assays for simultaneous

detection of analytes

Hanson,J.,Caspell,R.,Sundararaman,S.,Karulin,A.,Lehmann,P.

CellularTechnologyLtd,ShakerHeights,UnitedStates

ELISPOT assays are a key research tool for enumerating antigen-

specific T and B cells in PBMC. As both T and B cells occur in major

classes, immune monitoring has to be concerned with identifying

these as well. So far, ELISPOT assays have been primarily done single

or double color. We report the development of four color T and B

cell ELISPOT assays. We show that the four color assay has the same

sensitivity for detecting individual cells secreting analytes as the

respective single color assays, and that the four fluorescent colors

can be discerned unambiguously, without overlap. Cells secreting

any of the four analytes can therefore be identified unambiguously

in an automated fashion, without the need for compensation. Cells

co-expressing analytes can be identified by superimposing the

individual colors. Studying B cells and T cells experimentally has

permitted us to verify the accuracy of co-expressionmeasurements.

Each B cell secretes only one type of Ig class/subclass. T cells,

in contrast, frequently coexpress cytokines. Serial dilution

experiments showed that for T cells the numbers of co-expressors

linearly decreased with the numbers of cells plated. For B cells, no

coexpressors were found.

A positive control for the detection of functional CD4 T cells in

human PBMC - CPI protein pool

Schiller, A., Ansari, T., Li, R., Horvath, K., Sundararaman, S., Lehmann, P.

CellularTechnologyLtd,ShakerHeights,UnitedStates

Testing of PBMC for immune monitoring purposes requires

verification of the functionality. This is of particular concern when

the PBMC have been shipped or stored for prolonged periods

of time. The CEF peptide pool has become the gold standard for

testing CD8 cell functionality. A positive control for establishing

CD4 cell quality, that also requires intact antigen processing and

presentation functionality, is so far lacking. Protein antigens from

infectious/environmental organisms have been selected that are

ubiquitous. Of an initial selection of 12 antigenic systems, (Varicella,

Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus,

Mycoplasma,Lactobacillus,Neisseria,Candida,Rubella,andMeasles)

three were selected because they A) elicited CD4 cells exclusively,

and B) elicited recall responses in the majority of human donors.

These were inactivated Cytomegalo, Parainfluenza, and Influenza

virions.While individually none of the three antigens triggered recall

responses inall of the donors, the pool of these three antigens did. In

100 of 100 human donors (of different demographics) tested so far,

the CPI (Cytomegalo, Parainfluenza, and Influenza viruses) protein

pool triggeredapositive IFN-γ recall responsebyCD4 cells.

Therefore, theCPI proteinpool is suitableas apositive control for CD4

functionality.

Direct detection of T and B memory lymphocytes reveals

HCMV exposure that serum antibodies fail to identify

Caspell,R.

,Terlutter,F.

,Nowacki,T.

,Li,R.

,Sundararaman,S.

Lehmann,P.

CellularTechnologyLtd,ShakerHeights,UnitedStates,

UniversityhospitalofMünster,Munster,Germany

It is essential to identify donors who have not been infected with

HCMV in order to avoid transmission to human recipients of

transfusion products or of organs. In addition, since HCMV infection

hasbeenlinkedtothepathogenesisofvariousdiseases,itis important

tounambiguouslyestablishwhohas, andwhohasnot been infected.

In the present study, we tested the reliability of seronegativity as an

indicator for the lack of HCMV exposure of human subjects. Sixty six

HCMV seronegative individuals have been identified and their PBMC

were tested in ELISPOT assays for the presence of HCMV-specific

CD4, CD8 T - and B - memory lymphocytes. Fifty seven percent

of the HCMV seronegative subjects displayed CD4 and CD8 T cell

memory inaddition toHCMV specificmemoryBcellsproviding three

independent lines of evidence for having developed immunity to

HCMV. Fifteenpercent of the 66 seronegative donors possessed CD4

and CD8memory cells to HCMV, however, in the absence of memory

B cells, and 16% had CD4 memory cells in isolation. Only 12% of the

seronegative donors showed neither T- nor B- cell memory to HCMV

qualifying as immunologically naïve to the virus. The data suggest

that measurements of serum antibodies frequently fail to reveal

HCMV exposure of humans, whichmay be better identified by direct

detectionof HCMV-specificmemory lymphocytes.

Maximizing odds for detecting a positive T cell response by

ELISPOT

Roen,D.,Karulin,A.,Sundararaman,S.,Lehmann,P.

CellularTechnologyLtd,ShakerHeights,UnitedStates

It has been a matter of debate to determine the best cut offs in

ELISPOT assay analysis for the unambiguous identification of

a positive T cell response. To address this issue we carried out

experiments on HLA-A2-0201 positive human subjects who had

beeninfectedwithHCMV.However,weselecteddonorswhosePBMC,

when tested at 100,000 cells per well and challenged with the HLA-

A2-0201-restricted HCMV peptide, pp65(495-503) did not display

spot counts over medium background. Increasing the number of

PBMC plated per well resulted in higher positive to negative count

ratio, lower relative experimental error (CV), and higher power for

detecting pp65-induced positive responses without causing false

positive results from HCMV negative subjects. This decrease of CV

and increase in the power of the test was directly proportional to the

numbers PBMC plated. We tested the PBMC for pp65 reactivity in 96

replicate wells to establish the distributional properties of these low

frequency ELISPOTs. The results were analyzed using diagnostic Q-Q

plots and Shapiro-Wilk normality tests which showed that the spot

counts in replicate wells followNormal

distribution.We

showed that

parametric statistics, such as Student

-Test can be used and provide