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Therapeutic Day Schools for Middle and High School Students with Autism
Acacia Academy
La Grange Campus: 6425 Willow Springs Rd
Burr Ridge Campus: 6880 N. Frontage Rd Suite 100
info@acaciaacademy.com
Acacia Academy is an accredited, private special education / therapeutic school for elementary (grades 1-8), high school (grades 9-12) and Transition Program for ages 18-22. We offer a variety of school year and summer programs designed to meet each student's learning objectives in terms of his/her learning capabilities. Acacia Academy is AdvancED accredited and has been approved for out-of-district placement by the Illinois State Board of Education for special education. Students from all over northern Illinois attend Acacia Academy.
Academy of Excellence in Learning
19056 Henry Drive, Mokena, IL 60448
stephanie@academyofexcellenceinlearning.com and lindsay@academyofexcellenceinlearning.com
Illinois State Board of Education approved, non-public special education facility. They are currently accepting students with educational diagnosis of autism, cognitive disability, learning disabilities, challenging behaviors, speech or language impairment, and other health impairment.
Beard Special Education Center
6445 W. Strong St., Chicago, IL 60656
Offers education and treatment for autism spectrum, emotionally disabled, and cognitively delayed children including, discrete trial, sensory integration, behavioral, music, and art therapy, group theraplay and public talking circle.
Bi-County Special Education Cooperative
Admin Office: 1900 W. LeFevre Road, Ste. A Sterling, IL 61081
Services portions of Erie, Fulton, Prophetstown, Sterling, Mount Pleasant, Coloma, Fairhaven, and Freeport townships.
Bridge High School (Little Friends)
2318 Wisconsin Ave., Downers Grove, IL 60515
Therapeutic day school for children ages 14-22 with autism or other emotional, cognitive disabilities and emotions disorders that have experienced difficulty in traditional academic programs and require an alternative functional curriculum.
The Camelot Schools
Locations in Florida, Illinois, New Jersey, Pennsylvania, Texas & Virginia
info@cameloteducation.org
Illinois locations in Chicago, Custer Park, Garden Prairie, Genoa, Hoffman Estates, Moline, Mount Prospect and Naperville. Children participating in the autism program at Camelot's day schools take part in a specialized curriculum that focuses on maximizing independent functioning in home, school, vocational and community settings by utilizing music therapy, occupational therapy, speech/language therapy, social skills training, prevocational and vocational instruction, and a functional academic curriculum.
Cardinal Cushing Centers
405 Washington St. Hanover, MA 02339
mmarkowitz@cushingcenters.org
Private, non-profit therapeutic special needs boarding school for children and young adults with autism and developmental disabilities.
Chicago Autism Academy
21133 S. 80th Ave., Frankfort, IL 60423
CAACorwin@gmail.com
Serves children who have an autism spectrum disorder, cognitive disability, or a developmental delay with a variety of programs and empirical based procedures, which include: speech and language, occupational therapy, behavioral analysis, and social programming. A variety of empirically based procedures are used including, Behavior Analysis, Precision Teaching, Direct Instruction, and many other interventions. The Chicago Autism Academy accepts students from many school districts across Northeastern Illinois.
The Chicago Lighthouse for People Who Are Blind or Visually Impaired
mary.zabelski@chicagolighthouse.org
Therapeutic day school: Programs for students with blindness/visual impairments, moderate to severe/profound cognitive impairments, hearing impairments, traumatic brain injuries, autism, sensory processing disorders, seizure disorders, physical disabilities, communication disorders and other health concerns; program includes speech therapy, physical therapy, occupational therapy, social work, nursing, psychology, orientation and mobility, and vision and hearing.
Connections Day School
31410 North US Highway 45 | Libertyville, IL 60048
We serve children and adolescents, ages 8-21, primarily from Lake County who, because of significant special education needs (ED, BD, LD, OHI, AUT & CD), require an alternative school program that is both educational and therapeutic. By utilizing therapeutic, educational, family and community connections, CDS is able to promote academic achievement, social/emotional well-being and personal growth.
Connections South Campus
909 E. Wilmette Road, Palatine, IL 60074
South Campus is a private therapeutic day school serving students with a variety of emotional and behavioral needs serving students in Southern Lake and Northern Cook Counties who are in need of an alternative school program, which is supportive, therapeutic and educational. The program serves students between the ages of 6 and 21 years. SC utilizes educational, therapeutic, family and community connections to promote academic achievement and personal/emotional growth.
Easterseals Chicagoland & Greater Rockford
School locations in Chicago, Rockford and Tinley Park
Autism services include screening and diagnostics (ages 3-12), parent training, individual therapy, parent support groups, and resource support. Therapeutic day school programming available children ages 5-21.
Eisenhower Cooperative
Admin Office: 5318 W. 135th St. Crestwood, IL 60418
Services portions of Worth, Bremen, Thornton, and Calumet townships. The Eisenhower Cooperative provides special education services to children and consultative and professional development services to staff from the following ten elementary and high school districts in southern Cook County. Programs include:
CARE Program: Services elementary aged students in a therapeutic day school who exhibit emotional disturbances and severe emotional difficulties in their home school.
Deaf/Hard of Hearing Program: The Instructional Deaf and Hard of Hearing (DHH) Program utilizes a total communication philosophy to help students develop expressive and receptive language skills by offering opportunities for students to imitate, create, and expand their language base and usage.
Developmental Learning Program: Provides a complete educational program for students with multiple disabilities in a regular education setting. Children are with age-appropriate peers for lessons that are educationally appropriate to the individual child. Students in the Developmental Learning Program work on an individualized academic and functional curriculum and also have access to all related services included Speech/Language therapy, Occupational and Physical therapy on-site. Students also have access to a full-time nurse and assistive technology support as needed.
Elim Christian School
Autism Comprehensive Education (ACE) Program
13020 S. Central Ave., Palos Heights, IL 60463
School offers a one-on-one integrated holistic approach of services including TEACCH, applied behavior analysis, and augmentative communication.
Esperanza School
520 N Marshfield Chicago IL 60622
Ages: 6-21 years. Esperanza School is a private therapeutic day school that serves special education students with severe and profound Autism and Intellectual Disabilities, aged 6 through 21 years old. Due to their autism and other developmental disabilities, our students also display significant behavioral needs. Our multidisciplinary team includes teachers, behavior therapists, social workers, nurses, occupational therapists and a speech and communication specialist.
Genesee Lake School
Contact: Stephanie Koster-Peterson, admissions director
36100 Genesee Lake Road Oconomowoc, WI 53066
admissions@geneseelakeschool.com
Genesee Lake School is a nationally recognized provider offering programs for students with developmental disabilities, mental health disorders, neurological disorders, and emotional disturbances. Additionally, we offer specialized programs for students with autism spectrum disorders, mood and anxiety disorders, and complex trauma. Our 160 acre campus is a nurturing setting where students from around the country gain the skills and experiences necessary to successfully return to their families, school districts, and communities. Our goal at Genesee Lake School is to provide an innovative and enriching environment for the students in our care to learn and experience all that life can offer. Illinois State Board of Education Approved and COA Certified. Ages: 6-21 years.
Giant Steps Inc.
2500 Cabot Dr., Lisle, IL 60532
ssmith@mygiantsteps.org
Giant Steps Illinois day school offers a one-on-one integrated holistic approach of services for students with Autism Spectrum Disorders.
Grundy County Special Education Cooperative
Admin Office: 725 School St. Morris, IL 60450
GCSEC member districts service portions of Mazon, Morris, Aux Sable, Braceville, Garfield, Erienna, and Greenfield townships.
River's Edge Academy
725 School St Morris, IL 60450
Mr. Russell Young, Program Coordinator: ryoung@grundyspecialed.org
The Life Skills classrooms serve students with moderate to severe cognitive disabilities in grades K-12 at Rock Falls and Morrison Schools. Emphasis is placed on functional academics, daily living, self-care, communication, self-advocacy, and vocational skills appropriate to the age of the students.
Guiding Light Academy
1051 Internationale Pkwy, Woodridge, IL 60517
Woodridge, IL (Academy)- (630) 739-7500
Woodridge, IL (Central) - (630) 328-0438
Shorewood, IL (West) - (815) 409-4495
Guiding Light Academy accepts referrals for students from various school districts in the Chicagoland area. Potential students must be in grades K-12 and have a primary diagnosis of Autism, Other Health Impairment, Intellectual Disability, and Developmental Delay.
Helping Hand School for Autism
Sally Wilson-Gamble
9649 W. 55th St., Countryside, IL 60525
Use research-based intervention modalities and teaching methods to assure best practice to design each child's own individualized program and curriculum.
Holtz Educational Center
8149 Cass Ave., Darien, IL 60561
Holtz Educational Center, LLC is a non-public, therapeutic day school that provides special education alternatives to students pre-K through 8th grade utilizing the principles of Applied Behavior Analysis (ABA). Holtz accepts students diagnosed with Autism Spectrum Disorder, developmental delay, students previously diagnosed as having PDD-NOS, intellectual and specific learning disorders & other health impairments.
Jewish Child & Family Services
Joy Faith Knapp Children's Center
3145 W. Pratt Blvd, Chicago, IL 60645
therapeuticdayschool@jcfs.org
Provides the best educational, emotional and therapeutic services for students of all backgrounds in grades K-12 who have not thrived in a traditional school setting, to encourage their academic, interpersonal and vocational success.
Kankakee Area Special Education Cooperative (KASEC)
Admin Office: 650 W Guertin St, St Anne, IL 60964
KASEC member districts service portions of Aroma, Ashkum, Manteno, Saint Anne, Bourbonnais, and Pembroke townships. Programs include:
The Alternative Day Program, located at St. Anne Grade School and St. Anne High School, provides services and assistance to meet educational, social/emotional and behavioral needs of students from northern Iroquois and Kankakee Counties.
The Communications Program is for learners with significant speech-language, social-communication, or autism spectrum disorders to recognize and achieve their full potential. The Communications Program classroom is housed in public school building to maximize opportunities for learning and interaction with non-disabled peers.
Kemit Academy
5100 South Dorchester Avenue Chicago Illinois 60615
Kemit Academy offers specialized day care for infants and toddlers and a therapeutic day school for children ages 5-12 with developmental disabilities. Kemit also provides pediatric occupational and speech therapies.
Kradwell School
1220 Dewey Avenue Wauwatosa, WI 53213
A unique learning environment on Aurora Psychiatric Hospital's wooded campus. Kradwell is a private, nontraditional and nonsectarian school for children in grades 5 through 12. The school specializes in serving students who struggle to succeed in traditional school environments. The teachers and support staff develop educational plans tailored to each student and any conditions they may have, such as dyslexia, ADD/ADHD, anxiety, bipolar disorder, depression, PSTD, OCD, reactive attachment disorder, Asperger's syndrome and more. Contact form
Krejci Academy/The Mansion (Little Friends)
619 E Franklin Ave, Naperville, IL 60540
Therapeutic day school for children ages 5-21 with autism or other emotional, behavioral or developmental disabilities.
The LaGrange Area Department of Special Education
Admin Office: 1301 W Cossitt Ave La Grange, IL 60525
LADSE is a special education cooperative made up of 15 member districts (https://www.ladse.org/member-districts.html) in DuPage and Cook counties. LADSE collaborates with its member districts to provide high-quality, evidence-based programs and services and provide comprehensive, cost-efficient support, and positive outcomes for students with disabilities ages 3 through 22.
LEEP Quantum Leep Therapeutic School Program
Ravenswood (Chicago): 1447 W Montrose Ave.
Dana@leepforward.com
Offered a rolling enrollment K-8th Grade therapeutic school for individuals with autism. Call for details on tuition, annual fee varies based on daily therapeutic programming and insurance benefits. Afterschool programs available.
Leyden Area Special Education Cooperative (LASEC)
Admin Office: 10401 W Grand Ave Franklin Park, IL 60131
Services portions of Leyden Township elementary, middle, and high schools.
The Phoenix Academy is a public day school, middle school through transition, designed to provide an educational environment where youth can develop and implement skills to expand personal insight to improve their academic success, emotional health, and long term success in their community.
Lincoln-Way Area Special Education District 843
Admin Office: 601 Willow Street Frankfort, IL 60423
Services portions of Manhattan, Frankfort, and New Lenox townships.
Pioneer Grove Educational Center
601 Willow Street Frankfort, IL 60423
lwase@lwase843.org
The classes at Pioneer Grove Educational Center are designed to meet the unique needs of students from ages 3 to 21 including the following programs: Self-Contained Individualized Life Skills Program (SCILS), Supported Opportunities for Adapted and Life Skills Readiness (SOAR), Adapted Individualized Methods (AIM), and Multi-Needs.
Marklund
info@marklund.org
Locations in:
Bloomingdale: 64 S. Prairie Ave., Bloomingdale, IL 60108
Elgin: 1435 Summit Street, Elgin, Illinois 60120
Geneva: 1S450 Wyatt Drive, Geneva, IL 60134
Programs include Early Intervention services (Ages 0-3; 630-529-2871, ext. 2238), day school programming (Ages 3-21), day program services (Ages 18+) and residential services (all ages), and employment services for individuals with developmental disabilities.
The Manhattan Childrens Center (MCC)
111 West 92nd Street, New York, NY 10025
The Manhattan Children's Center (MCC) is a not-for-profit, 501(c)(3) school for Autism education and treatment based in New York City. MCC provides world-class treatment and education to children with autism spectrum disorder (ASD).
Mid-Valley Special Education Cooperative Organization
Admin Office: 1304 Ronzheimer Avenue St. Charles, IL 60174
Services students with special needs in portions of Hanover, Burlington, Kaneland, Geneva, and St. Charles townships.
The Mades-Johnstone Center (MJC), in St. Charles, houses administration and a small number of programs. MVSEC also rents a portion of the Shelby Building and Baker Memorial Church in St. Charles and Fox Valley Christian Church in Batavia that house our Transition Program. Most programs and services are located in school districts.
New Connections Academy
New Connections Academy serves boys and girls ages 6 to 21 with ASD placed by local school districts. New Connections Academy provides school, family and community partnerships leading to successful learning. New Connections Academy offers a primary program (grades 1 to 3), intermediate (grades 4-6), junior high (grades 7 & 8) and several high school classes (grades 9-12). The classes are self-contained, offering a standard elementary curriculum of Reading, Spelling, Language Arts, Mathematics, Science, Social Studies, and Physical Education. Each student has an Individual Educational Plan developed by the placing district.
New Hope Academy
Brandy Larrance, executive director
6289 W. Howard St. Niles IL 60714
Illinois State Board of Education approved. Serves students in special education diagnosed with emotional disabilities, other health impairments, learning disabilities, and autism. Students are average to above-average intelligence, withdraw socially and emotionally, experience difficulty "fitting in" a mainstream environment, have difficulty reading social cues, "slip through the cracks" until their problems are severe, procrastinate (specifically with homework assignments and long-term projects), have difficulty with executive functioning, have talents in art and music, have the potential for academic achievement, struggle with gross and fine motor skills, have difficulty with social skills, use frequent illness as a means to avoid school and may become "school phobic," appear sad, lonely, hopeless, or depressed, harbor anger and hostility but does not act out violently in school or in the community. Ages: 11-21 years.
New Horizon Center
6737 W. Forest Preserve Ave, Chicago, IL 60634-1470
Patricia Palmer: ppalmer@newhorizoncenter.org
Therapeutic day school for those with severe and profound learning disabilities, autism, and intellectually challenged students who benefit from a personalized program with services such as speech therapy, physical therapy, occupational therapy, music therapy, art therapy, nursing services, social services, vision services, sensory room, Rainbow Dog Therapy, Greenhouse Gardening and much more.
North DuPage Special Education Cooperative
132 E. Pine Avenue Roselle, IL 60172
Services portions of Addison, Leyden, and Bloomingdale townships.
NDSEC assists its nine member districts (https://www.ndsec.org/organization) with the provision of special education instructional and related services. NDSEC operates a variety of programs designed to address the needs of its students, maintaining a full continuum of placement options in three programs: Itinerants, Academic Life Skills, and Social Support.
Northwest Suburban Special Education Organization
Admin Office: 799 W. Kensington Rd., Mount Prospect, IL 60056
Services provided in both public and therapeutic day schools throughout Niles Township.
Provides specialized services including professional learning, adapted physical education, assistive and instructional technology, evaluation and coaching, vision services, transition services, autism support, and deaf & hard of hearing evaluation services. NSSEO offers programming for diverse learners at three therapeutic day school campuses and D/HH programs at the elementary, middle, and high school levels within district public schools.
O-School (The Sonia Shankman Orthogenic School at the University of Chicago)
Therapeutic Day School Program
Kristin Friesen, LCSW (Director of Admissions)
6245 S. Ingleside Ave., Chicago, IL 60637
The O-School is a therapeutic day school for individuals with high functioning autism and/or other mental health issues. The O-School is Illinois State Board of Education approved. The day school is committed to fostering inquiry into the clinical and treatment needs of students. The O-School relies upon the therapeutic relationships that develop between a student and their teachers and therapists as a major component of the treatment. Milieu staff members are actively involved with the students throughout the day, not simply monitoring or supervising them but developing bonds and relationships with students that create a structure in which each child or adolescent can receive emotional support, develop new skills and sustain healthy relationships. The school and family work together to create a consistent experience for each student. Serves ages: 5-21; Funded through private pay or child's school district.
PACTT Learning Center
7101 N. Greenview Ave., Chicago, IL 60626
learningcenter@pactt.org
Illinois State Board of Education approved. PACTT is an agency that provides educational, residential and vocational services to individuals with autism.
Proviso Area For Exceptional Children
Admin Office: 1000 Van Buren St. Maywood, IL 60153
Proviso Area for Exceptional Children provides services for member districts that service parts of Stone Park, Berkeley, Northlake, Bellwood, Maywood, Melrose Park, Broadview, Westchester, Forest Park, Elmhurst and Hillside. Programs include:
PAEC Center Program provides programs designed to address the needs of special education students ranging in age from three years old to the day before their 22nd birthday. The programs include a range of interventions for students with autism, multiple disabilities and cognitive disabilities.
The Cross Categorical Instructional Program at PAEC Academy is for students with mild to moderate disabilities K-8.
The Early Childhood Program provides special education preschool classes to children between the ages of 3-5 who have developmental delays. Students receive related services including social work, speech and language therapy, occupational therapy, physical therapy, vision itinerant services, and/or other related services.
The PAEC Therapeutic Day Alternative School environment has been developed to provide students k-12 diagnosed with severe social/emotional disabilities with intensive, supportive and individualized mental health services in addition to opportunities for academic success.
Rush Day School
Jean L. Heideman, MPP, MS, director
2150 W. Harrison St. Chicago, IL 60612
Illinois State Board of Education approved. Rush Day School utilizes a team approach to provide intensive psychotherapeutic services combined with an academic program. Offers education and treatment for autism spectrum and emotionally disabled children. Goal for each student is to return to a less restrictive school environment. The majority of students return to a public or private school in regular education with support. Class size: limited to six to nine students. Children may be enrolled throughout the academic and extended school year. Ages: 5-15 years (grades K-8).
The School Association for Special Education in DuPage County
Admin Office: 2900 Ogden Ave Lisle, Illinois 60532
SASED is the fiscal agent for DuPage West Cook Regional Education Association (DWC) who provides low incidence special education programs and services to students in the identified catchment area (https://www.sased.org/who-we-are/member-districts). SASED is a regional provider of Low Incidence programs and services for students who are Blind/Visually Impaired or Deaf and Hard of Hearing. There are 92 school districts and cooperatives in DuPage and West Cook Counties that are a part of this regional cooperative that include CASE, NDSEC, and LADSE.
Soaring Eagle Academy
8330 S. Madison St., Burr Ridge, IL 60527
info@soaringeagleacademy.org
First school in the Midwest to provide a social and academic learning environment utilizing a developmental individual-difference relationship based (DIR) approach for students diagnosed with autism and related disorders.
Special Education District of Lake County
Admin Office: 18160 W Gages Lake Rd Gages Lake, IL 60030
Services portions of Benton, Waukegan, Zion, Newport, Warren, Antioch, Grant, Avon, Libertyville, Moraine, Shields, Vernon, West Deerfield, Fremont, Wheeling, Lake Villa, and Wauconda townships.
SEDOL services Lake County via Sector, Itinerant, and Transition programs to 31 Lake County member districts with physical, emotional, and other learning challenges. SEDOL also operates eight therapeutic day schools for elementary, middle, high school, and transition-aged students.
Special Education Joint Agreement District 802
Admin Office: 1125 Division Street Chicago Heights, IL 60411
Services portions of Bremen, Bloom, and Rich townships.
Provides several specialized in-district programs and day schools for elementary, middle, high school, and transition students.
Stuart G. Ferst School
6050 N. California Ave., Chicago, IL 60659
Illinois State Board of Education approved. Provides co-ed functional academic instruction, vocational experience and/or therapeutic education to students with behavioral/emotional disorders, traumatic brain injuries, or developmental disabilities such as autism, cognitive impairment or physical/medical impairment.
Summit School
The Autism Spectrum Program
333 W. River Road, Elgin, IL 60123
jwhite@summitdundee.org
Program offers a one-on-one integrated holistic approach of services including DIR, TEACCH and RDI.
Thome ED School
500 East Fifth Street Rock Falls, IL 61071
Thome ED School is a K-12 school that provides special education services for children who experience difficulty in their home schools due to severe emotional challenges. Small class sizes and a wide range of support services allow students to improve their ability to function within the classroom setting, with the goal of returning to their home schools with both academic and socially appropriate behaviors.
301 Veterans Parkway New Lenox, IL 60451
Trinity School was founded in 1950, and has evolved to Trinity Services, Inc. to provide a broad range of supports and services for people with disabilities. The school remains a vital support for students with intellectual and developmental disabilities between the ages of 3 and 21. Issues may include developmental delays, autism, behavioral challenges and attention deficit disorder.
True North Educational Cooperative 804
Admin Office: 760 Red Oak Lane Highland Park, IL 60035
True North (previously Northern Suburban Special Education District (NSSED)) has 18 member districts, fourteen elementary and four high school that service the northern suburbs of Chicago out of both Lake and Cook counties. NSSED provides a wide array of programs and services designed to give students access to the highest levels of learning possible. Informed by research and policy, NSSED and member districts have historically believed that diverse learners are best served when they learn alongside their typical peers. NSSED programs and services partner with member districts to increase inclusive opportunities that promote high expectations, engagement, and growth for all learners.
Turning Pointe CN Day School
1500 W. Ogden Ave. Naperville, IL 60540
bbassett@turningpointeaf.org
Turning Pointe's therapeutic day school program serves middle and high school students impacted by autism. Our trademark COPES (Communication, Organization, Positive Behavior Supports, Emotional Regulation, Sensory and Social) method is utilized to help students progress. Programming is highly individualized as outlined in students' Individual Education Plans (IEP) and includes a collaborative therapeutic team approach. Additional services may include: Speech and Language, Occupational Therapy, Behavior Analysis, Dog Therapy, and Recreational Leisure. Students practice life and vocational skills to increase their independence. These skills are generalized into the community setting. Ages: Middle School, High School, and Transition
The Winston Knolls School
2353 Hassell Rd, Suite 110, Hoffman Estates, IL 60169
info@knollscenterforautism.org
Services Provided: Provides ABA Therapy, Occupational Therapy, Speech Language Pathology, Physical Therapy, Social Skills Groups, Parent Training and Education, Family Counseling, Social Work, Psychiatry, and Academic Programs through insurance.
Provides interdisciplinary pediatric therapy and support for the whole child, and your whole family. Services include: Early Intervention, Feeding therapy, Speech Therapy, Physical Therapy, Occupational Therapy and Counseling. Additional autism services include ABA, DIR/Floortime, assistive technology clinics, adaptive swim lessons, care management, diagnostic evaluations, social skills groups, residential day camp and parent training. Therapeutic day school programming available for children ages 5-21.
ppalmer@newhorizoncenter.org
Linda Cervenka


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text:
package org.jpox.samples.interfaces;

/**
 * Rectangle class.
 **/
public class Shape5Rectangle implements Shape5
{
	private int id;
    protected double width = 0.0;
    protected double length = 0.0;

    protected Shape5Holder shapeHolder = null;

    public Shape5Rectangle()
    {
    }

    public Shape5Rectangle(int id, double width,double length)
    {
        this.id = id;
        this.length = length;
        this.width = width;
    }

    public String getName()
    {
        return "Rectangle";
    }

    public void setLength(double length)
    {
        this.length = length;
    }

    public void setWidth(double width)
    {
        this.width = width;
    }

    public double getArea()
    {
        return length*width;
    }

    public int getId()
    {
        return id;
    }

    public void setId(int id)
    {
        this.id = id;
    }

    public double getLength()
    {
        return length;
    }

    public double getWidth()
    {
        return width;
    }

    public Shape5Holder getShapeHolder()
    {
        return shapeHolder;
    }

    public boolean equals(Object o)
    {
        if (o == null || !o.getClass().equals(this.getClass()))
        {
            return false;
        }
        Shape5Rectangle rhs = (Shape5Rectangle)o;
        return width == rhs.width && length == rhs.length;
    }

    public String toString()
    {
        return "{Rectangle width=" + width + "; length=" + length + "}";
    }
}

redpajama_set_name: RedPajamaGithub


text:
The Size and Phenotype of Virus-Specific T Cell Populations Is Determined by Repetitive Antigenic Stimulation and Environmental Cytokines
Laila E. Gamadia, Ester M. M. van Leeuwen, Ester B. M. Remmerswaal, Si-La Yong, Sugianto Surachno, Pauline M. E. Wertheim-van Dillen, Ineke J. M. ten Berge and René A. W. van Lier
J Immunol May 15, 2004, 172 (10) 6107-6114; DOI: https://doi.org/10.4049/jimmunol.172.10.6107
Laila E. Gamadia
Renal Transplant Unit, Department of Internal Medicine, Laboratory for Experimental Immunology,
Ester M. M. van Leeuwen
Ester B. M. Remmerswaal
Laboratory for Experimental Immunology,
Si-La Yong
Sugianto Surachno
Renal Transplant Unit, Department of Internal Medicine,
Pauline M. E. Wertheim-van Dillen
Department of Virology, and
Ineke J. M. ten Berge
Renal Transplant Unit, Department of Internal Medicine, Division of Clinical Immunology and Rheumatology, Department of Internal Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
René A. W. van Lier
Based on the expression of the TNFR SFP CD27, two Ag-primed CD8+ T cell subsets can be discerned in the circulation of healthy individuals: CD27+ T cells that produce a variety of cytokines but do not display immediate cytolytic activity; and cytotoxic CD27− T cells, which secrete only IFN-γ and TNF-α. The mechanism that controls the generation of these different phenotypes is unknown. We show that CMV reactivation not only increases the number of virus-specific T cells but also induces their transition from a CD27+ to a CD27− phenotype. In support of a relation between pool size and phenotype in a cohort of latently infected individuals, the number of Ag-specific CD27− CD8+ T cells was found to be linearly related to the total number of CMV-specific CD8+ T cells. In vitro studies revealed that the acquisition of the CD27− phenotype on CMV-specific T cells depended on the interaction of CD27 with its cellular ligand, CD70. Expression of CD70 was proportional to the amount of antigenic stimulation and blocked by the CD4+ T cell-derived cytokine IL-21. Thus, induction of CD70, which may vary in distinct viral infections, appears to be a key factor in determining the size and phenotype of the CMV-specific T cell population in latently infected individuals.
T cell memory is established after clearance of acute infection and maintained lifelong. The generation of CD8+ memory T cells is supposedly by the survival of a number of Ag-stimulated effector T cells that are generated early in the primary adaptive immune response to the causative pathogen. What determines a primary induced CD8+ T lymphocyte to survive into a long-lived memory cell is as yet unclear (1). Primed T cells in the peripheral blood of healthy individuals show considerable phenotypic and functional heterogeneity (2). At one side of the spectrum are CD45R0+CD28+CD27+ T cells that produce a large variety of cytokines, but are unable to execute cytolysis without prior in vitro culture (3). These cells have been defined as memory-type T cells because of their ability to clonally expand in response to recall Ags in the absence of helper T cell-derived cytokines. Based on the expression of the chemokine receptor CCR7, this population may be subdivided into CCR7+ central memory T cells that have the ability to migrate to secondary lymphoid organs and CCR7− effector memory T cells that do not have this ability (4). It is unclear whether CCR7+ and CCR7− T cells are also functionally distinct (5, 6). Separate from these memory-type cells is a population of CD45RA+CCR7−CD28−CD27− T cells (further referred to as CD45RA+CD27− T cells). Based on the expression of CD95 ligand, perforin, and granzyme B and the ability to execute cytotoxicity directly ex vivo, these T cells have been designated effector-type T cells. This population, which is also CD11ahigh and CD57+, increases with age and has a restricted diversity of TCR-Vβ genes (7, 8, 9).
T cells with specificities for different persistent viruses vary in phenotype and function. For instance, EBV-specific T cells are predominantly CD45R0+CD28+CD27+, and in asymptomatic HIV-carriers, HIV-specific T cells are mostly CD45R0+CD28−CD27+, contrasting the phenotype of CMV-reactive T cells that are predominantly CD45RA+CD27− (10). From these findings, it was postulated that viruses might induce phenotypically distinct T cells (10). However, it is clear that the selection for phenotypes is not absolute because, e.g., in healthy CMV carriers during latency, virus-specific T cells with either CD45R0+CD27+ and CD45RA+CD27− surface profiles can be found (10, 11). Analysis of virus-specific T cells during acute infections with EBV, HIV, or CMV revealed that early effector T cells are clearly distinct from the effector-type CD45RA+CD27− T cells found in latently infected persons. T cells expanding early in immune responses have CD45R0, CD28, CD27 surface expression, express the G1 phase-associated nuclear Ag Ki-67, and contain perforin and granzyme B (12, 13, 14). These observations raised questions about the generation of CD45RA+CD27− T cells and their role in protective immunity (10).
Although it was originally proposed that CD45RA+CD28−CD27− T cells had entered a state of senescence (15), telomere length analyses revealed similar replicative histories in vivo of CD45R0+CD27+ and CD45RA+CD27− CD8+ T cells (8). Moreover, it was shown that CD45RA+CD27− T cells were unable to divide when stimulatory mAbs to CD3 and CD28 are combined to activate these cells (2). Still, CMV-specific T cells with this phenotype potently expanded when stimulated by specific MHC/peptide ligands in the presence of helper T cell-derived cytokines (16, 17). These latter experiments implied that although CD45RA+CD27− T cells are fully differentiated T cells where it concerns CTL effector functions, they are not terminally differentiated (18) in the sense of having lost the ability to yield progeny.
Here, we studied patients with well-documented CMV reactivation to investigate the generation of CD27− T cells in vivo and their role in maintaining CMV latency. We show that effector-type T cells can expand in vivo when viral replication occurs. Moreover, antigenic load and helper T cell-derived cytokines are key regulators of the phenotype of virus-specific T cells during latency, likely through regulation of the CD27 ligand, CD70.
Five HLA-A2-positive and 1 HLA-B7-positive CMV-seropositive renal transplant recipients of a kidney of either a CMV-seropositive donor (2) or a CMV-seronegative donor (4) were longitudinally studied. Basic immunosuppressive therapy consisted of cyclosporin A, blood trough level aimed at 150 ng/ml, mycophenolate mofetil (1000 mg twice daily), and prednisone (10 mg daily). Viral replication was monitored longitudinally by quantitative PCR from the time point of transplantation, and reactivation was determined by detectable viral loads above the cutoff point of 80 copies/ml. All subjects experienced asymptomatic CMV reactivation and no antiviral treatment was given. Heparinized peripheral blood samples were collected before transplantation and weekly during 17 wk after transplantation, whereafter samples were collected once a month. PBMC were isolated using standard density gradient centrifugation techniques and subsequently cryopreserved. In addition, CMV-specific T cells were characterized in 11 healthy individuals and 36 renal transplant recipients on basic immunosuppressive therapy either before or after reactivation as measured by PCR. All patients gave written informed consent, and the local medical ethical committee approved the study.
The HLA-A2-binding CMV pp65-derived peptide NLVPMVATV and the HLA-B7-binding CMV pp65-derived peptide TPRVTGGGAM were purchased from the IHB-LUMC peptide synthesis library facility (Department of Immunohaematology and Blood Bank, Leids Universitair Medisch Centrum, Leiden, The Netherlands).
Generation of tetrameric complexes
Tetrameric complexes were manufactured at the tetramer facility of Sanquin, The Netherlands, and generated essentially as described by Altman et al. (19) In brief, purified HLA-A2.1 H chain or HLA-B7.2 H chain and β2-microglobulin were synthesized using a prokaryotic expression system (pET; Novagen, Milwaukee, WI). The H chain was modified by deletion of the transmembrane-cytosolic tail and C-terminal addition of a sequence containing the BirA enzymatic biotinylation site. The HLA-A2.1 binding CMV pp65-derived peptide NLVPMVATV and the HLA-B7.2-derived peptide TPRVTGGGAM were used for refolding. Monomeric complexes were concentrated, biotinylated by BirA (expressed using the pET expression system, and purified using Clontech cobalt beads; Clontech Laboratories, Palo Alto, CA) in the presence of biotin (Molecular Probes, Eugene, OR), ATP (Sigma-Aldrich, St. Louis, MO), and MgC12. The biotinylated product was separated from free biotin by fast protein liquid chromatography using a Superdex 200 HR16/60 column (Amersham Pharmacia, Little Chalfont, U.K.). Streptavidin-allophycocyanin conjugate (Molecular Probes) was added in a 1:4 molar ratio, and subsequently tetramers were fast protein liquid chromatography purified using the same column.
CFSE labeling
PBMCs were pelleted and resuspended in PBS at a final concentration of 5–10 × 106 cells/ml. PBMCs were labeled with 2.5 μM CFSE (Molecular Probes) in PBS for 8 min, shaking at 37°C. Cells were washed and subsequently resuspended in IMDM supplemented with 10% human pool serum, antibiotics, and 3.57 × 10−4 % (v/v) 2-ME (Merck, West Point, PA; culture medium).
Culture and stimulation of cells
CFSE-labeled cells were cultured in culture medium for 5 days in 24-well plates at a concentration of 0.5–1 × 106 cells/ml. CMV pp65-derived peptide was added at a final concentration of 1.25 μg/ml. CMV-Ag (inactivated whole virus, 10 μl/ml; Microbix Biosystems, Toronto, Canada) was used to stimulate cells. Furthermore, for stimulation, IL-2 (50 U/ml; Biotest, Dreieich, Germany), IL-15 (3 ng/ml; R&D Systems, Abingdon, U.K.), and IL-21 (50 ng/ml; Zymogenetics, Seattle, WA), were added. For blocking experiments, we used CD70 Ab (clone 2F2; Ref.20) at concentrations of 40 μg/ml. For dose-dependent analysis of CD70 expression, 1.25 μg of peptide and 10 μl of CMV-Ag/ml were titrated. Flow cytometric analysis was performed before culture and after 5 days.
Immunofluorescent staining and flow cytometry of CMV-specific CD8+ T cells
Thawed PBMC were resuspended in RPMI containing 10% FCS and antibiotics; 200,000 PBMC were incubated with fluorescent label-conjugated mAbs (concentrations according to manufacturer's instructions) and an appropriate concentration of tetrameric complexes. Negative controls to validate specificity of the CMV-peptide-tetrameric complexes consisted of HLA-A2.1/HLAB7.2-negative CMV-seropositive or HLA-A2.1/HLA-B7.2-positive CMV-seronegative healthy individuals and renal transplant recipients. Negative controls always showed tetramer staining of <0.01% of total lymphocytes (data not shown). For staining with the mouse anti-human CCR7 mAb, a three-step staining protocol was performed consisting of incubation with the CCR7 Ab (BD PharMingen, San Diego, CA), for 30 min, washing, incubation with biotinylated goat anti-mouse IgM (BD PharMingen) for 30 min, and incubation with 10% (v/v) normal mouse serum (Sanquin, Amsterdam, The Netherlands) followed by incubation with streptavidin-PE and directly conjugated mAbs and tetrameric complexes for 30 min. For staining with the mouse anti-human mAb CD70 (clone 2F2) a two-step staining protocol was performed consisting of incubation with the CD70 Ab for 30 min, washing, incubation with FITC-conjugated anti-mouse IgG1 Abs, washing, and incubation with 10% (v/v) normal mouse serum followed by incubation of directly conjugated mAbs. Analyses consisted of allophycocyanin-conjugated tetramers and CD8-PerCP (BD Biosciences, San Jose, CA) in combination with either CD28 (Sanquin) and CD27 (BD Biosciences), CCR7 and CD45RA, CD27 and CD45RA (BD Biosciences) and CD45RA and CD45R0 (BD Biosciences), CD38 (BD Biosciences), and CD70, all combinations in FITC and PE, and additional stainings for CD70 expression were done with CD70 in combination with CD4-PE, CD8-allophycocyanin and CD19-PerCP.
CMV-PCR
Quantitative PCR was performed in EDTA whole blood samples as described for plasma or serum (21).
Linear regression analysis using a mixed model was performed for the absolute numbers of total CMV-specific CD8+ T cells in relation with total CD27−CMV-specific CD8+ T cells, and for the percentage CMV-specific CD8+ T cells and the percentage of CD27− of CMV-specific CD8+ T cells taken as a logit. Subsequently, the regression coefficient was calculated and tested for significance. Repeated observations were analyzed by paired Student's t test or a repeated measures ANOVA; p values < 0.05 were considered statistically significant.
CMV reactivation induces quantitative and qualitative changes in the CD8+ virus-specific T cell compartment
In healthy individuals and renal transplant recipients during latency, CMV-specific CD8+ T cells may have different predominant phenotypes ranging from CD45R0+CD28+CD27+ (memory-type, patient 1; Fig. 1⇓a; t = 0) to CD45RA+CD45R0dull/−CD27− (effector-type, patient 2; Fig. 1⇓d; t = 0) (10, 11). The fate of these distinct CMV-specific T cell populations was followed during CMV reactivation.
Quantitative and qualitative changes of CMV-specific CD8+ memory cells during reactivation. a–c, Differentiation of CMV-specific CD8+ T cells in one patient with a starting population of CD45R0+CD27+CD28+ CMV-specific cells. d–f, Differentiation of CMV-specific CD8+ T cells in one patient with a starting population of CD45RA+CD27−CD28− cells. Time defined as t = 0 before reactivation, t = 1 during reactivation at peak PCR value, and t = 2 at first time point after reactivation. All plots gated on CD8+ T cells. CMV-specific CD8+ T cells as defined by specific tetramer staining plotted in black; total CD8+ T cells are plotted in gray. Percentage of CMV-specific CD8+ T cells of patients 1 and 2, respectively, at time points 0–2: Patient 1, 0.23, 0.42, and 2.55%; patient 2, 2.21, 2.83, and 2.96%. a and d: x-axis log fluorescence CD27-FITC; y-axis log fluorescence CD28-PE; percentages depicted are the percentage of CD27− cells of total tetramer+ cells. b and e: x-axis log fluorescence CD45R0-PE; y-axis log fluorescence CD45RA-FITC; c and f: x-axis log fluorescence CD38-PE; y-axis log fluorescence HLA-DR-FITC.
In the patients with a CD45RA−CD28+CD27+ CD8+ T cell phenotype before reactivation, a substantial number of these cells lost expression of CD28 and CD27 (Fig. 1⇑, row a). Longitudinal analysis of CMV-specific CD8+CD27− T cells in these individuals showed that this subset increased immediately when replication of virus occurs (Fig. 2⇓a, CD27−tetramer+ T cells in relation to viral load in one representative individual; Fig. 2⇓b, CD27−tetramer+ T cells in all individuals; median before reactivation, 26.5%; range, 0.0 to 91.1%; median at the time point of reactivation, 45.22%) and keeps on doing so long after viral replication has ceased (median at the first time point after reactivation, 66.35%; median at the last time point measured, >6 mo after reactivation, 72.73%; p = 0.039). In the individuals with a predominant effector, CD45RA+CD28−CD27− CD8+ T cell phenotype, there was an increase in the number of CMV-specific T cells but no phenotypic change of virus-specific CD8+ T cells with regard to the expression of CD28 and CD27 (Fig. 1⇑, row d). To analyze whether this down-regulation of CD27 was not contributable to other factors as immunosuppressive medication upon transplantation, we also analyzed EBV-specific cells in individuals who were CMV and EBV seropositive, experiencing CMV reactivation and not EBV reactivation. As is shown in Fig. 2⇓c, CMV-specific cells increase in CD28−CD27−CD45RA+ cells, whereas EBV-specific cells, that in the individual shown were CD45R0+CD28+CD27+, do not change in surface phenotype after CMV reactivation, implying that this phenomenon is solely due to specific stimulation of CMV-specific cells.
Ongoing down-modulation of CD27 on CMV-specific CD8+ T cells after reactivation. a, Longitudinal analysis of CD27 expression on CMV-specific CD8+ T cells in relation to CMV viral load in one representative patient with a CD28+CD27+CD45R0+ phenotype of CMV-specific cells before CMV reactivation. ▾, viral load; •, number of CD27− CMV-specific CD8+ T cells. b, Percentage of CD27− CMV-tetramer+ T cells in all patients during follow-up of reactivation. Time defined as: t = 0, before reactivation; t = 1, during reactivation at peak PCR value; t = 2, first time point after reactivation; and t = 3, >6 mo after reactivation. c, Expression of CD27, CD28, CD45RA, and CD45R0 on CMV-specific cells (left) and EBV-specific cells (right) before and 1 year after CMV reactivation in two representative patients. All plots gated on total tetramer+ T cells.
Irrespective of the prereactivation phenotype, the activation markers CD38 and HLA-DR were up-regulated on CMV-specific cells in all patients at the time of viral replication (Fig. 1⇑, rows c and f), as was the expression of CD45R0, reflecting recent proliferation of these cells (Fig. 1⇑, rows b and e). CCR7 expression, documented to be up-regulated upon activation (17, 18), did not change in any of the patients during reactivation (data not shown), although re-expression of this marker could redirect subjected cells to secondary lymphoid organs, rendering them undetectable in peripheral blood.
These data suggest that irrespective of their phenotype, CMV-specific T cells can confer protective immunity in situations of virus reactivation. Moreover, virus reactivation appears not only to increase the size of the virus-specific T cell pool but also, in individuals that start off with memory-type CMV-specific T cells, to induce a change from a predominant CD27+ to a CD27− phenotype.
The CD27− phenotype is correlated with the magnitude of the CD8+ T cell response during latency
The above findings suggested a connection between the number of virus-specific T cells in latency and their phenotype which would accord with the expansion of CMV-specific CD8+CD27− T cells in immunocompromised patients (11, 22), B cell chronic lymphocytic leukemia patients (23), and the elderly (24, 25).
Indeed, close analysis of the total percentage of CD8+ CMV-specific cells as determined by tetramer staining and the expression of CD27 by these cells shows a strong correlation between the loss of CD27 and a higher percentage of CMV-specific CD8+ T cells as determined by tetramer staining, for both renal transplant recipients and healthy control individuals. The correlation of the percentage of CMV-tetramer+CD8+ T cells and the percentage of CD27− of these cells is depicted in Fig. 3⇓a, which shows that after the percentage of tetramer+ cells reaches ∼1%, ≥50% of these cells are CD27−, whereas virtually all CMV-specific cells are CD27− when a percentage of 2% or higher is reached. Also, when depicted in absolute numbers, the amount of CMV-specific cells is linearly correlated to the amount of CD27− cells (Fig. 3⇓b).
High frequencies of CMV-specific CD27− effector cells are correlated to the total amount of CMV-specific CD8+ T cells. a, Correlation of the percentage of total CMV-tetramer+ T cells and the percentage of CD27− of CMV-specific T cells. •, Values in renal transplant recipients before or after reactivation; ○, values in healthy control individuals; curve, functional relationship determined by the formula % CD27− = 100 ∗ [(e**(0.226 + 0.86*ln(% tetramer))/(1 + (e**(0.226 + 0.86*ln(% tetramer))]; p = 0.005. b, Correlation of the absolute number of total CMV-tetramer+ T cells and the absolute number CD27− CMV-specific T cells. •, Values in renal transplant recipients; curve, linear relationship determined by absolute number of CMV-tetramer+ cells = 0.001 + (1.46 ∗ absolute number of CMV-tetramer+CD27−) (p = 0.0012).
Generation of the CD27− phenotype of virus-specific T cells is dependent on interaction of CD27 with its cellular ligand, CD70
T cells down-modulate CD27 after interaction with its cellular ligand CD70 both in vitro and in vivo (26, 27). In this process, T cells receive costimulatory signals for expansion (26, 27) and acquisition of effector functions (28). To analyze whether virus-induced CD70 expression could account for the down-modulation of CD27 on virus-specific T cells after CMV reactivation, CD70 expression was measured in the reactivating patients. Indeed, whereas during latency most CMV tetramer-binding T cells were CD70 negative (Fig. 4⇓, top left), CD70 transiently increased during reactivation episodes (Fig. 4⇓. middle and right). Accordingly, viral Ag-induced CD70 expression could be induced in vitro, and the magnitude of CD70 expression showed a dose-response relationship with the virus-derived stimulus (Fig. 4⇓, bottom).
CD70 expression is induced by Ag and is dose dependent. a, CD70 is expressed on CD8+ and CMV-specific CD8+ T lymphocytes during viral replication in vivo. All plots gated on CD8+ T cells. Time defined as: t = 0, before reactivation; t = 1, during reactivation; and t = 2, after reactivation. Quadrant percentages depicted as percentage CD70+ of tetramer+ cells. b: left, Upon in vitro stimulation with CMV-Ag and CMV-peptide, CD70 expression is induced on CD8+ T cells. x-Axis log fluorescence CFSE; y-axis CD70 expression, gated on CD8+ T cells; total CD8+ T cells plotted in gray, and CMV-specific CD8+ T cells plotted in black. Right, Expression levels of CD70 on CD8+ T cells in various doses of CMV-Ag and peptide stimulation. x-Axis log fluorescence CD70; filled histogram, no stimulus; closed histogram, stimulation with 0.15625 μl/ml CMV-Ag and 0.0195 μg/ml CMV-peptide; dotted histogram, stimulation with 0.625 μl/ml CMV-Ag and 0.0781 μg/ml CMV-peptide; bold histogram, stimulation with 2.5 μl/ml CMV-Ag and 0.3125 μg/ml CMV-peptide.
To test whether viral Ag-induced up-regulation of CD70 dictated CD27 expression, T cells from donors with a CD45R0+CD27+ phenotype (Fig. 5⇓a) were cultured for 5 days with specific peptide and CMV-Ag to activate helper T cells in the absence and presence of blocking CD70 mAb. In these cultures, CMV-specific T cells rapidly down-regulated CD27 (Fig. 5⇓b). However, addition of CD70 mAb to these cultures reversed this blocking effect and yielded CD27+ CMV-specific T cells at day 5 of culture. Irrespective of CD27 down-regulation, these cells remained CD28+. The observation that CD28 is down-regulated in vivo during CMV reactivation implies that other signals induce CD28 down-regulation. Helper CD4+ T cells can directly support CD8+ T cell expansion through secretion of stimulatory cytokines and provision of membrane-bound helper factors (29). Furthermore, the homeostatic cytokine IL-15 was shown to be involved in maintenance and activation/functional differentiation of memory CD8+ T cells (30, 31, 32). To analyze a possible qualitative role of different cytokines on CD8+ T cell differentiation, the effects of cytokines, which previously were found to support proliferation of CMV-specific T cells (17), on CD70 and CD27 expression were analyzed. In conjunction with peptide, IL-2 and IL-15 induced CD70 expression on CMV-specific T cells and CD70-dependent down-modulation of CD27 (Fig. 6⇓, a and b). Although IL-21 was reported to stimulate only CD45RA+ thymocytes (33), in our experiments, in an Ag-specific setting, CD45R0+CD8+ T cells were also responsive to IL-21. Interestingly, IL-21, a cytokine presumed to limit effector cell expansion (34), did not induce CD70 expression and supported propagation of CD27+ CMV-specific T cells without differentiation (Fig. 6⇓c).
CD70 induces differentiation of CD27+ CMV-specific CD8+ T cells. a, Expression of CD70 on total lymphocytes from the donor shown in Fig. 1⇑a after stimulation with CMV-peptide and CMV-Ag (open histogram) and no stimulus (filled histogram). x-Axis log fluorescence CD70. b, Histogram overlay of CD27 expression on tetramer+CD8+ T cells after 5 days of stimulation with peptide and CMV-Ag with or without blocking of CD70. CD27 expression on CMV-specific CD8+ T cells without blocking of CD70 (filled histogram) and with blocking of CD70 (open histogram). c, Histogram overlay of CD28 expression on tetramer+CD8+ T cells after 5 days of stimulation with peptide and CMV-Ag with or without blocking of CD70. CD28 expression on CMV-specific CD8+ T cells without blocking of CD70 (filled histogram) and with blocking of CD70 (open histogram). One representative donor of four tested is shown.
CD70 expression is induced by IL-2 and IL-15 but not by IL-21. Left, Expression of CD70 on total lymphocytes after stimulation with CMV-peptide and IL-2, IL-15, or IL-21, respectively (open histogram) and no stimulus (filled histogram). x-Axis log fluorescence CD70. Right, Dot plots of CD27 expression on tetramer+CD8+ T cells after 5 days of stimulation with peptide and IL-2, IL-15, or IL-21, respectively, with or without blocking of CD70. Plots gated on CD8+ T cells. x-Axis log fluorescence CMV-tetramer; y-axis log fluorescence CD27. Histogram overlays of CD27 expression on CMV-specific CD8+ T cells with different stimulations without blocking of CD70 (filled histogram) and with blocking of CD70 (open histogram). One representative donor of four tested is shown.
Our results show that in persistent viral infection, reactivation increases the number of memory cells and skews these cells to a CD27− phenotype. In primary infection, the amount of Ag determines the size of the murine CD8+ T cell response. Subsequently, the magnitude of this CD8+ T cell response sets the size of the Ag-specific T cell pool during the memory phase (35, 36). Recently, it was demonstrated that the contraction phase of the primary immune response is independent of the clearance of Ag (37). However, both cytokines present during initial priming and the amount of Ag present after the contraction phase are likely to have an impact on the quality and quantity of the memory cells rendered thereafter (36, 37, 38). It was proposed that different viruses elicit different human CD8+ T cell responses with respect to their surface phenotype (10). EBV and HIV predominantly elicit CD28−/+CD27+CD8+ responses. The analysis of Ag-specific memory CD8+ T cells directed against influenza virus (39, 40), which is completely cleared from the host, reveals that these cells are all CD28+CD27+ and present at low percentages compared with the frequency of CD8+ T cells directed at persistent viruses. CMV-specific responses in both healthy individuals and renal transplant recipients vary in magnitude and phenotype among individuals (10, 11, 16). The strong correlation between the percentage of virus-specific cells in the total CD8+ T cell population and the percentage of CD27− cells of the virus-specific cells indicates that a uniform mechanism takes place that drives both the expansion of virus-specific memory cells and their differentiation to cytotoxic effector-type cells.
In this study, replication of CMV, measured by PCR, could be pinpointed to a very limited period shortly after transplantation, whereas the increase in total CMV-specific cells and the down-modulation of CD27 on these cells was an ongoing process for up to >1 year after transplantation. The ongoing rise in CMV-specific CD8+ T cells therefore is possibly generated by an increase in the number of infected host cells that persistently present Ag to immune cells. The only known mechanism through which CD27 is down-regulated is after interaction with its ligand CD70. CD70 belongs to the TNF family, and its expression on B and T cells can be induced by antigenic stimulation in vitro (41) and in vivo (this study) and appears to be modulated by the cytokine milieu. We do not know at this moment where the interaction between CD27 and CD70, leading to the down-modulation of the former molecule, takes place in vivo during CMV reactivation. Moreover, although we showed that virus-specific T cells can express CD70, we also found that B cells and CD4+ T cells in patients with positive CMV PCRs express considerable amounts of CD70 (data not shown) and therefore may have the ability to trigger and modulate CD27 on CMV-reactive T cells. Our study showed that TCR triggering in combination with IL-2 and IL-15 induced CD70, whereas IL-21 did not give CD70 expression. Accordingly, IL-2 and IL-15 induced expansion of CD27− virus-specific T cells, whereas IL-21 promoted outgrowth of CD27+ cells. Although both IL-2 and IL-21 are helper cell-derived cytokines, the differentiation of CMV-specific memory CD8+ T cells to a CD27− phenotype upon activation with whole CMV-Ag in combination with peptide shows that the role of IL-21 production by CMV-specific CD4+ memory T cells, reported to have a Th1 profile (42), is limited in accordance with previous reports in which IL-21 was shown to be a Th2 cytokine (43). Whereas production of helper cell-derived cytokines as IL-21 and IL-2 is limited to the antigenic activation phase, IL-15 can be produced by a number of lymphoid and nonlymphoid cells (44). It is feasible that viruses elicit distinct helper cell cytokine profiles by differential infection of APC and by different target cell tropism, thereby also determining the number and phenotype of CD8+ T cells during the memory phase (45, 46, 47).
CD27-deficient mice have a reduced ability to form adequate numbers of Ag-specific T cells upon viral infection (48). In contrast, mice that constitutively express CD70 on B cells have a strongly enhanced capacity to form effector T cells in response to viruses, tumors, and protein Ags (Ref.49 and R. Arens, manuscript in preparation). Thus, these mouse models strongly infer that the CD27-CD70 interaction is a major determinant in setting the size of the Ag-specific pool after immunization. The way in which CD27 signaling regulates these processes is incompletely unraveled, but CD27−/− T cells show diminished survival after stimulation (48). An effect of CD27 on T cell survival would be in line with proposed functions of related TNFR family molecules such as OX40 and 4-1BB that increase expression of antiapoptotic molecules such as Bcl-2 and Bcl-xL (50, 51, 52). Indeed, recently we found strong up-regulation of Bcl-xL expression by CD27 triggering (M. van Oosterwijk, manuscript in preparation). On the basis of these observations, it can be postulated that through pathogen-induced expression of CD70 and subsequent triggering of CD27, up-regulation of Bcl-xL and increased survival of Ag-specific T cells, the contraction phase after initial expansion is altered leading to a higher setpoint in memory cell numbers. Simultaneously, through prolonged survival, T cells may receive differentiation signals for a longer period. This proposed link between survival and differentiation may explain the clear correlation we found between CMV-specific T cell numbers and their phenotype. In our in vitro studies, we did not find an effect of CD70 blockade on numbers of Ag-specific T cells, although these Abs were capable of blocking CD27 signaling (Figs. 5⇑ and 6⇑). The lack of effect might be explained by the fact that up-regulation of Bcl-xL may protect T cells from death by neglect, i.e., cytokine shortage, which may be essential in vivo but irrelevant in our cultures where ample cytokines are present.
Taken together, our data imply that the induction and expansion of CD8+ virus-specific T cells and their differentiation to the CD27− effector phenotype is driven by both antigenic load and helper T cell-derived or homeostatic cytokines. We submit that regulation of CD70 expression could be central in this process. Elucidating the differentials inducing and sustaining the CD27− effector phenotype could have a profound impact on immunotherapeutic strategies, especially in disease situations in which strong and persistent cytolytic responses are warranted.
We thank Dr. G. J. Weverling for expert statistical analysis, Dr. D. van Baarle for expert technical assistance regarding tetramer manufacturing, and Dr. F. Miedema, Dr. E. Eldering, and Dr. R. Arens for critical reading of the manuscript.
↵1 This work was supported by Dutch Kidney Foundation Grant C98-1724 (to L.E.G. and E.B.R.).
↵2 Address correspondence and reprint requests to Dr. Rene A.W. van Lier, Academic Medical Centre, University of Amsterdam, L1-152, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands. E-mail address: r.vanlier{at}amc.uva.nl
Copyright © 2004 by The American Association of Immunologists
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Vol. 172, Issue 10
You are going to email the following The Size and Phenotype of Virus-Specific T Cell Populations Is Determined by Repetitive Antigenic Stimulation and Environmental Cytokines
Laila E. Gamadia, Ester M. M. van Leeuwen, Ester B. M. Remmerswaal, Si-La Yong, Sugianto Surachno, Pauline M. E. Wertheim-van Dillen, Ineke J. M. ten Berge, René A. W. van Lier
The Journal of Immunology May 15, 2004, 172 (10) 6107-6114; DOI: 10.4049/jimmunol.172.10.6107
Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION

redpajama_set_name: RedPajamaCommonCrawl


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