title: Optimization of in vitro expansion of macaque CD4 T cells using anti-CD3 and co-stimulation for autotransfusion therapy.
abstract: BACKGROUND
  Our laboratory has previously shown that adoptive transfer of in vitro-expanded autologous purified polyclonal CD4(+) T cells using anti-CD3/CD28-coated beads induced antiviral responses capable of controlling SIV replication in vivo.
  
  
  METHODS
  As CD4(+) T cells comprise several phenotypic and functional lineages, studies were carried out to optimize the in vitro culture conditions for maximal CD4(+) T-cell expansion, survival and delineate the phenotype of these expanded CD4(+) T cells to be linked to maximal clinical benefit.
  
  
  CONCLUSIONS
  The results showed that whereas anti-monkey CD3gamma/epsilon was able to induce T-cell proliferation and expansion in combination with antibodies against multiple co-stimulatory molecules, monkey CD3epsilon cross reacting antibodies failed to induce proliferation of macaque CD4(+) T cells. Among co-stimulatory signals, anti-CD28 stimulation was consistently superior to anti-4-1BB, CD27 or ICOS while the use of anti-CD154 failed to deliver a detectable proliferation signal. Increasing the relative anti-CD28 co-stimulatory signal relative to anti-CD3 provided a modest enhancement of expansion. Additional strategies for optimization included attempts to neutralize free radicals, enhancement of glucose uptake by T cells or addition of T-cell stimulatory cytokines. However, none of these strategies provided any detectable proliferative advantage. Addition of 10 autologous irradiated feeder cells/expanding T cell provided some enhancement of expansion; however, given the high numbers of T cell needed, this approach was deemed impractical and costly, and lower ratios of feeder to expanding T cells failed to provide such benefit. The most critical parameter for efficient expansion of purified CD4(+) T cells from multiple monkeys was the optimization of space and culture conditions at culture inception. Finally, anti-CD3/28-expanded CD4(+) T cells uniformly exhibited a central memory phenotype, absence of CCR5 expression, marked CXCR4 expression in vitro, low levels of caspase 3 but also of Bcl-2 expression.
authors.affiliation: Department of Pathology and Lab Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Nattawat
authors.initials: N
authors.lastname: Onlamoon
authors.forename: Krystal
authors.initials: K
authors.lastname: Hudson
authors.forename: Patsy
authors.initials: P
authors.lastname: Bryan
authors.forename: Ann E
authors.initials: AE
authors.lastname: Mayne
authors.forename: Mark
authors.initials: M
authors.lastname: Bonyhadi
authors.forename: Ron
authors.initials: R
authors.lastname: Berenson
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Sundstrom
authors.forename: Pavel
authors.initials: P
authors.lastname: Bostik
authors.forename: Aftab A
authors.initials: AA
authors.lastname: Ansari
authors.forename: François
authors.initials: F
authors.lastname: Villinger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D048708:Cell Growth Processes / Q000276:immunology; D003429:Cross Reactions; D016207:Cytokines / Q000276:immunology / Q000494:pharmacology; D004195:Disease Models, Animal; D005434:Flow Cytometry; D016130:Immunophenotyping; D016219:Immunotherapy, Adoptive / Q000379:methods; D008213:Lymphocyte Activation; D008253:Macaca mulatta / Q000276:immunology*; D016097:Simian Acquired Immunodeficiency Syndrome / Q000276:immunology / Q000628:therapy; D015302:Simian Immunodeficiency Virus / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D017252:CD3 Complex; D016207:Cytokines