Presenter Disclosure Information Presenter Disclosure Information Michael P. Gustafson, Ph.D. The...

Presenter Disclosure InformationPresenter Disclosure Information

Michael P. Gustafson, Ph.D.

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LOSS OF HLALOSS OF HLA--DR EXPRESSION ON CD14+ CELLS; A DR EXPRESSION ON CD14+ CELLS; A COMMON MARKER OF IMMUNOSUPPRESSION IN COMMON MARKER OF IMMUNOSUPPRESSION IN

CANCER PATIENTSCANCER PATIENTS

Michael P. Gustafson, Ph.D.Michael P. Gustafson, Ph.D.

Human Cellular Therapy LaboratoryHuman Cellular Therapy LaboratoryMayo ClinicMayo Clinic

2010 2010 iSBTC iSBTC Annual MeetingAnnual Meeting

Assessing patient immunity by peripheral Assessing patient immunity by peripheral blood blood immunophenotypingimmunophenotyping

● Flow Cytometry● Whole blood● Broad unbiased approach

> 40 phenotypes● Patients: prior to therapy or

> 8 weeks off treatment

DiseasesGlioma Stage IV (GBM) n=50

Prostate Cancer n= 40Non-Hodgkin’s Lymphoma (NHL) n=40

Clear Cell Renal Cell Carcinoma (RCC) n=23Chronic Lymphocytic Leukemia (CLL) n=29

Patients at risk for sepsis n=29Diabetes and ALS n=11

TOTAL=193 patients

LymphocytesT cells

T helper/T killer subsetsT regulatory cellsCostimulatory/inhibitor receptorsActivated T cellsCentral and effector memoryNaïve T cells

B cellsNaïve and Mature B cellsTransitional B cellsPlasma cellsIsotyped switched

NK cellsNKT cells

Myeloid cellsMonocytes

M1 and M2 (CD14+ vs CD16+) HLA-DRCo-stimulatory receptorsSignaling receptors

GranulocytesCD15+CD66b+

Myeloid Derived Suppressor cellsClassical Lin-HLA-DR-CD33+“Granulocytic” CD15+CD14-

Generating Generating ““Immune ProfilesImmune Profiles”” using using bioinformaticsbioinformatics

Phenotypes converted to cells/uL

Data analyzed using Partek

Each sphere representing individual immune profiles

Clustering identifies relationships

Provides ‘unbiased’method to look for phenotypes of interest

CD14CD14++HLAHLA--DRDRlo/neglo/neg monocytes profoundly monocytes profoundly change the immune profile of patient change the immune profile of patient PBMCsPBMCs

NKBCD4CD8TregCD14+Dr+CD14+Dr-MDSCs

NKBCD4CD8TregCD14+Dr+CD14+Dr-MDSCs

Control

GBM

CD14+

DR+

DRlo/neg

CD14+

DR+

DRlo/neg

With enough disease profiles, average profile of patients can bedetermined

CD14CD14++HLAHLA--DRDRlo/neglo/neg monocytes are elevated in monocytes are elevated in cancer patientscancer patients

Normal Donor Patient

CD14CD14++HLAHLA--DRDRlo/neglo/neg monocytes mechanisms of monocytes mechanisms of immunosuppressionimmunosuppression

Gustafson MP, Lin Y, et al, Neuro-Onc. 2010; 12:631-44.Vuk-pavlovic, et al, Prostate 2010; 70: 443-55.Lin Y, Gustafson MP, et al, Submitted manuscript.

Inhibition of T cell proliferation Unable to fully differentiate into mature DCs

CD14CD14++HLAHLA--DRDRlo/neglo/neg in other clinical settingsin other clinical settings

Found in melanoma, ovarian Found in melanoma, ovarian cancer, and cancer, and hepatocellular hepatocellular carcinoma.carcinoma.

Valenti Valenti R, Cancer Res. 2006; R, Cancer Res. 2006;

Loercher Loercher AE, J. AE, J. ImmunolImmunol. 1999; . 1999;

Hoechst B, Gastroenterology, 2008.Hoechst B, Gastroenterology, 2008.

Associated with sepsis, acute Associated with sepsis, acute pancreatitis, liver failure, burns, pancreatitis, liver failure, burns, and trauma.and trauma.

Correlated with survival in Correlated with survival in sepsis.sepsis.

Cheadle Cheadle WG, Am. J. Surg. 1993; Wakefield CH, et al, WG, Am. J. Surg. 1993; Wakefield CH, et al, Br. J. Surg. 1993; van den Br. J. Surg. 1993; van den Berk Berk JM, et al, JM, et al, Transplantation 1997; Transplantation 1997; Lekkou Lekkou A, et al, A, et al, ClinClin. . Diag. Lab. Diag. Lab. ImmunolImmunol. 2004.. 2004.

Loss of HLALoss of HLA--DR on CD14+ cells is a DR on CD14+ cells is a prognostic factor in cancer patientsprognostic factor in cancer patients

Normal HLA-DR + 1 other phenotypeAbnormal HLA-DR + 1 other phenotype

GBM NHLN=40

N=25

SummarySummary

Immunophenotyping Immunophenotyping by flow by flow cytometry cytometry and and multiparameter multiparameter analysis will continue to be extremely analysis will continue to be extremely important in characterized baseline immunity in patients. important in characterized baseline immunity in patients.

Bioinformatics approach is likely to yield new relationships Bioinformatics approach is likely to yield new relationships between immune cells.between immune cells.

CD14CD14++HLAHLA--DRDRlo/neglo/neg monocytes are elevated in every monocytes are elevated in every cancer type that wecancer type that we’’ve analyzed.ve analyzed.

CD14CD14++HLAHLA--DRDRlo/neglo/neg monocytes inhibit T cell proliferation monocytes inhibit T cell proliferation and cannot fully mature into potent and cannot fully mature into potent DCsDCs..

The combination of CD14The combination of CD14++HLAHLA--DRDRlo/neglo/neg monocytes and monocytes and other phenotypes are prognostic; independent of therapy. other phenotypes are prognostic; independent of therapy.

ImplicationsImplications

The presence of CD14The presence of CD14++HLAHLA--DRDRlo/neglo/neg monocytes monocytes may identify potential responders/nonmay identify potential responders/non--responders responders on patients receiving vaccines or other on patients receiving vaccines or other immunotherapeutic approaches.immunotherapeutic approaches.

Mechanisms of Mechanisms of immunosuppression/immunosuppression/

immunoparalysis immunoparalysis are very similar in cancer are very similar in cancer patients and in infection/sepsis patients.patients and in infection/sepsis patients.

AcknowledgmentsAcknowledgments

Dr. Allan DietzDr. Allan Dietz Scientific DirectorScientific Director

Dr. Dennis GastineauDr. Dennis Gastineau Medical DirectorMedical Director

Peggy Peggy BulurBulur Mary MaasMary Maas

Other collaborators:Other collaborators: Dr. Yi LinDr. Yi Lin Dr. Dr. RoshiniRoshini AbrahamAbraham Dr. Dr. StanimirStanimir VukVuk--PavlovicPavlovic Dr Eugene KwonDr Eugene Kwon Dr. Clive Dr. Clive ZentZent Dr. Dr. JannJann SarkariaSarkaria Dr. Brian ODr. Brian O’’NeillNeill Dr. Ian Dr. Ian ParneyParney Dr. Kent NewDr. Kent New Dr. Thomas Dr. Thomas WitzigWitzig

Human Cellular Therapy Lab